The application of BacMam technology in nuclear receptor drug discovery

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The application of BacMam technology in nuclear receptor drug discovery Mohamed Boudjelal1, Sarah J. Mason1, Roy M. Katso3, Jonathan M. Fleming1, Janet H. Parham2, J. Patrick Condreay2, Raymond V. Merrihew4, and William J. Cairns1,* 1 Department of Gene Expression and Protein Biochemistry, Discovery Research Biology, GlaxoSmithKline Discovery Research, Harlow, UK 2 Research Triangle Park, NC, USA 3 Department of Assay Development and Compound Profiling, Discovery Research Biology, GlaxoSmithKline, Stevenage, UK 4 Research Triangle Park, NC, USA

Abstract. The nuclear receptor (NR) superfamily represents a major class of drug targets for the pharmaceutical industry. Strategies for the development of novel, more selective and safer compounds aimed at these receptors are now emerging. Reporter assays have been used routinely for the identification and characterisation of NR ligands. As the NR drug development process evolves, the increase in screening demand in terms of both capacity and complexity has necessitated the development of novel assay formats with increased throughput and flexibility. BacMam technology, a modified baculovirus system for over-expressing genes of interest in mammalian cells has helped answer this requirement. BacMam has many advantages over traditional gene delivery systems including high transduction efficiencies, broad cell host range, speed, cost and ease of generation and use. As outlined in this review, the technology has shown itself to be robust and efficient in various NR assay formats including transactivation (ERa/b, MR, PR and PXR) and transrepression (GR-NFkB). In addition, the flexibility of this system will allow greater multiplexing of receptor, reporter, and cell host combinations as NR assays become more complex in order to relate better to relevant cellular and biological systems. Keywords: Baculovirus, BacMam, nuclear receptor, cofactor, cell based assay, steroid receptor, pregnane X receptor, transient transfection.

Introduction Nuclear receptors (NRs) represent one of the most important families of drug targets for the pharmaceutical industry. They are transcription factors whose function can be regulated upon binding their cognate pharmacological ligands. Analysis of the entire human genome confirmed the existence of 48 NRs which had previously been identified using traditional molecular biology, biochemistry, genetic and bioinformatic techniques. The NR superfamily share two major conserved structural features (Fig. 1A). The first is a centrally located highly conserved DNA binding domain (DBD). This domain is typified by the presence of two conserved zinc finger modules that target the receptor to specific DNA *Corresponding author: Tel: +44 1279 622043. Fax: +44 1279 627666. E-mail: [email protected] BIOTECHNOLOGY ANNUAL REVIEW VOLUME 11 ISSN: 1387-2656 DOI: 10.1016/S1387-2656(05)11003-5

ß 2005 ELSEVIER B.V. ALL RIGHTS RESERVED

102 A N-Terminal Domain

N

DNA Binding Domain (DBD)

Ligand Binding Domain (LBD)

AF2

AF1

C

+ ligand Co-repressor

Co-activator

No ligand

B ERE

(G/A)GGTCA nnn TGACCT(T/C)

GRE, PRE, ARE, MRE

GGTACA nnn TGTTCT

PXR

AGTTCA nnn AGTTCA

Fig. 1. (A) Schematic representation of the domain structure of a typical nuclear receptor. Typically NRs are composed of an N-terminal domain that contains a ligand independent activation domain (AF1), followed by a zinc-finger DNA-binding domain and ligand binding domain (LBD). The LBD includes the ligand dependent activation domain (AF2). Upon binding of an agonist the co-repressor protein dissociates from the LBD and is replaced by the co-activator molecule. (B) The consensus palindromic DNA sequences of the response elements for the steroid receptor family and a PXR direct repeat response element are shown. The non-conserved intervening sequences are marked as ‘‘n’’.

sequences referred to as nuclear receptor responsive elements (some examples are shown in Fig. 1B). The steroid receptor subfamily all bind to palindromic sequences as homodimers, whereas most of the NR superfamily bind to direct repeat sequences spaced by 0–5 nucleotides as heterodimers with retinoic X receptor (RXR). Several receptors have also been shown to bind to half-sites as monomers (for a review see [1]). The second conserved domain in NRs is the C-terminal ligand binding domain (LBD) that is also known to play a role in dimerisation and transactivation. The NR superfamily has been divided into six subfamilies and 26 further subgroups based on evolutionary conservation [2]. To date, ligands have been identified for about half of the family while the rest remain as orphans. Research on NRs has brought forth advances in the treatment of inflammation, cancer, osteoporosis, and several endocrine and reproductive disorders. For example, a significant fraction of the most frequently prescribed drugs target the steroid receptors and peroxisome proliferating activating receptor-gamma (PPARg). Many pharmaceutical and biotech companies as well as academic laboratories are trying to identify natural or surrogate ligands for the remaining

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orphan receptors, while in parallel attempting to develop safer and more selective compounds for the liganded receptors [1]. Nuclear receptors represent one of the most complex target classes in drug discovery. They are expressed in a broad range of tissues and mediate diverse roles in physiological pathways. The discovery of novel NR selective compounds is a complicated and challenging process. Upon identification of a tractable target, a lead generation strategy is typically defined by an initial high throughput screen (HTS) (the ‘‘primary assay’’), followed by hit confirmation (‘‘secondary screening’’) and subsequently lead validation and optimisation. The purpose of high throughput screening is the interrogation of large and diverse chemical collections in the context of a biological target to accurately identify active chemotypes. The increase in size of the chemical compound libraries and the number of druggable targets obligate the development of new assay formats that can meet the elevated screening demand. Many factors can influence the assay format(s) employed in lead generation as well as their position in the screening paradigm. These include the type of pharmacological information required, throughput, cost and other logistical or practical considerations. Future nuclear receptor (NR) assays may involve complex interactions between receptors, cofactors, response elements and other cellular factors. Such conditions cannot be adequately reproduced with conventional in vitro biochemical assays. The importance of screening these targets in a cellular system in which all of the necessary components are present may therefore be paramount [3,4]. In this chapter we give a general overview of cell based reporter assay systems that are currently used in the discovery and characterisation of nuclear receptor ligands. We also describe the use of recombinant baculovirus technology for transduction of mammalian cells (BacMam) which has been employed for nuclear receptor cell based assays and shows multiple benefits over current transient transfection methods. Reporter assays in nuclear receptor drug discovery When an agonist binds to a nuclear receptor it initiates a series of events culminating in either transactivation or transrepression. Transactivation involves the recruitment of co-activators to form complexes which ultimately result in the stimulation of target promoter activity. Transrepression can occur directly by the NR binding to DNA and interfering with the activity of other transcription factors. Alternatively, it can be independent of DNA binding, being mediated via protein–protein interactions with other transcription factors. The regulation of the basal transcription machinery is believed to be mediated through a number of classes of cofactors (both co-activators and co-repressors) [5,6]. The ultimate effect of these processes is the induction or repression of target gene transcription. A variety of assay formats have been used for the identification and characterisation of new nuclear receptor ligands including in vitro NR ligand

104 binding assays and cell based reporter assays. Radiolabelled ligand-binding assays, such as scintillation proximity assays, measure the binding affinity of the ligand to the receptor [7]. Cell-based reporter assays measure the effect of the ligand on receptor activity indirectly through a reporter gene product. Reporter gene assays allow the discrimination between agonists, inverse agonists and antagonists, which cannot be determined in ligand binding assays alone. In addition, cell based reporter assays provide additional information such as the ability of the compounds to permeate the cell membrane. These characteristics make reporter assays an attractive format for nuclear receptor screening campaigns.

General principles of the reporter assay The key component of any reporter assay is a plasmid consisting of a quantifiable reporter gene fused to the regulatory region from an NR target gene. The reporter construct is delivered into cells by transient transfection, where its expression can then be regulated by either endogenous or exogenously expressed receptor upon the addition of ligand. Consequently, reporter assays can provide an insight into the mode of action of test compounds. The implementation of reporter assays in nuclear receptor drug discovery has been very successful. In addition, their widespread use has driven the development of multiple instrumentation platforms and commercial reagents that have been optimised to provide greater signal amplitude and stability. The development of low volume liquid handling automation has enabled reporter assays to be miniaturised into high density formats such as 384- and 1536-well plates [8]. There are three important components that should be integrated into the reporter construct: the reporter gene itself, a minimal promoter (containing the TATA box to which general transcription factors bind), and the transcriptional regulatory sequence that confers nuclear receptor-dependent regulation of the promoter (Fig. 2). The most frequently used reporter genes include chloramphenicol acetyl transferase (CAT), luciferase (Luc), b-galactosidase (b-GAL), b-lactamase (BLA), and secreted placental alkaline phosphatase (SPAP). The natural promoter of NR responsive genes can be used in the reporter plasmid thereby generating a highly relevant reporter construct. However, the regulatory sequences of NR responsive genes can lie far upstream from the start of transcription. In addition, the use of completely homologous promoters can result in elements other than the nuclear receptor binding site being present. In this case, the possible effects of other signalling pathways should be taken into consideration. Instead of a long promoter region containing a complex array of various elements, defined nuclear receptor regulatory elements can be inserted upstream of the reporter gene but fused to a heterologous promoter. The promoter from the herpes simplex virus thymidine kinase (HSV-tk) gene is

105 Ligand/Drug

or Full length NR

Gal4-NR

NRE or UAS

Promoter

Reporter gene

Reporter Plasmid

Fig. 2. Composition of a standard reporter plasmid. Schematic representation of a reporter plasmid based on a nuclear receptor response element (NRE) or Gal4 DNA binding element (UAS). When an NRE is used expression of full-length receptor in the presence of agonist is needed to induce reporter gene expression. When the more generic UAS-reporter plasmid is used, a Gal4-NR LBD chimera is employed in the assay. The NRE or UAS is placed upstream of a promoter sequence which drives reporter gene expression.

frequently used for this purpose. The NR responsive elements that are inserted are often multimerised in order to potentiate any response. An alternative reporter assay format that has been employed extensively uses a chimeric receptor by fusing the NR ligand binding domain (LBD) downstream of a heterologous DNA-binding domain such as that from the yeast Gal4 transactivator (Fig. 2). A reporter construct regulated via Gal4-DNA binding sites can then be used to measure activity. The Gal4 system has the advantage of being generic in that it allows multiple nuclear receptors to be screened in a single assay format. However, a major disadvantage of this approach is the use of a Gal4-LBD fusion rather than full length receptors. Genes used in reporter assays The selection of reporter gene systems for use in high throughput screening and other drug discovery related functions is often complex and depends on a number of factors. As mentioned previously, the most commonly used reporter genes in nuclear receptor research include luciferase (Luc) and secreted placental alkaline phosphatase (SPAP). Chloramphenicol acetyl transferase (CAT) has also been used historically while b-lactamase (BLA) is a relatively new format.

106 Table 1. Key features of the reporter genes commonly used in nuclear receptor reporter assays. Luc: Luciferase, SPAP: secreted placental alkaline phosphatase, CAT: chloramphenicol acetyl transferase, BLA: b-lactamase.

Reporter gene enzyme amplification Signal and sensitivity Adaptation to HTS Penetration of living cells Development of stable cell lines

Luc

SPAP

CAT

BLA

+++ +++ ++ + +

+++ ++ + +++ +

+++ +




+++ +++ +++ +++ +++

Table 1 compares the characteristics of these reporter genes and their utility in drug discovery. Luciferase Firefly luciferase is a 61kD monomeric protein that catalyses the monooxygenation of the substrate D-luciferin. In the presence of D-luciferin, O2 and ATP, an enzyme-bound luciferyl-adenylate complex is formed, followed by oxidative decarboxylation with the production of CO2, oxyluciferin, AMP and light [9]. Luciferase expression in mammalian cells has typically been measured by luminometer analysis of cell lysates upon addition of substrates and ATP. One of the newer reporter genes being used is the luciferase gene from Renilla reniformis (sea pansy). Renilla luciferase, a monomeric 36kDa protein, catalyses coelenterate-luciferin (coelenterazine) oxidation to produce light [10]. Secreted placental alkaline phosphatase assay (SPAP) The SPAP reporter gene encodes a truncated form of the placental enzyme lacking the membrane anchoring domain. This allows the protein to be efficiently secreted from transfected cells [11]. The enzyme is very stable, and no posttranslational modifications are necessary for enzymatic activity. Endogenously expressed alkaline phosphatase is not secreted and thereby does not interfere with reporter signal. Several colorimetric assays can be used, the most sensitive ones being Phospha-Light and Tropix which are comparable in expense and signal duration to luciferase. Unlike luciferase, SPAP is secreted which allows multiple measurements to be made easily from a single culture as cell lysis is not necessary for detection. Chloramphenicol acetyl transferase (CAT) Chloramphenicol acetyl transferase (CAT) is a bacterial drug-resistance enzyme that inactivates chloramphenicol by acetylating it at one or both of its two hydroxyl groups [12]. It is a stable enzyme that is not found in eukaryotes, and

107

therefore eukaryotic cells contain no background CAT activity. CAT activity can be assayed by liquid scintillation counting (LSC) of CAT reaction products. Alternatively the reaction products can be analysed using thin-layer chromatography (TLC). Although this method is more time consuming than the LSC assay it does allow visual confirmation of the data. Recently, an enzyme-linked immunosorbent assay (ELISA) for CAT has been developed which has made the use of this reporter gene somewhat more straightforward. However, even with this new development, the assay is laborious, expensive, and relatively insensitive. These characteristics also make the assay difficult to automate for high throughput screening. b-Lactamase Bacterial b-lactamase (Bla) has emerged as a useful reporter enzyme for gene expression assays due to the discovery of a novel fluorogenic substrate CCF2/ AM [13]. In the absence of b-lactamase reporter activity, the substrate molecule remains intact. Excitation of the coumarin at 409 nm results in fluorescence resonance energy transfer (FRET) to the fluorescein moiety. This energy transfer causes the fluorescein to emit a green fluorescence signal with an emission peak of 520 nm. In the presence of b-lactamase reporter activity, the CCF2 substrate is cleaved, disrupting FRET. In this case, excitation of the coumarin at 409 nm results in emission of a blue fluorescence signal with an emission peak of 447 nm. The detection sensitivity offered by Bla is ideal for high throughput assay formats. Major advantages of b-lactamase in HTS assays over other reporters include its fluorescence ratiometric readout, its high sensitivity and its reproducibility [14]. BacMam technology in nuclear receptor drug discovery Numerous chemical and physical gene delivery systems have been developed to introduce nuclear receptor and reporter DNA constructs into cells for reporter assays. Calcium precipitation and lipofection based transfection techniques have been used routinely for several years. However, the transfection efficiency can be extremely low for some cell types, while the process can be cytotoxic to others. Stable cell line generation has also been employed to provide reagents for screening, however this approach is extremely time consuming and labour intensive. The search for more efficient, less expensive techniques for overexpressing genes of interest in mammalian cells is ongoing in pharmaceutical and academic research laboratories. The use of virus based technologies as alternative gene-delivery tools has been under development for some time. Viruses are naturally equipped to penetrate host cells allowing overexpression of the genes that they are carrying in target cells. In particular, baculoviruses derived from the Autographa californica nuclear polyhedrosis virus (AcMNPV) have been used for many years

108 to overexpress recombinant proteins. The baculovirus system allows expression of the target gene in the AcMNPV natural host lepidopteran cells, commonly Spodoptera frugiperda cell lines. Deletion of the polyhedrin gene means that only the budding form of the virus is released rather than the occluded form ensuring intact host organisms cannot be infected. This minimises safety concerns when working with these viruses. Genes of interest can then be placed downstream of the polyhedrin promoter region for expression in insect cells [15]. Many features of the baculovirus system make it an attractive approach for the introduction and over-expression of genes in mammalian cells. It was proposed that modified baculoviruses containing genes under the control of mammalian cell promoters could deliver and drive overexpression of those genes in mammalian cells. In the mid 1990s this theory was confirmed by two independent groups who showed that such modified baculoviruses could transduce mammalian cells and mediate over-expression of the genes they carried. These studies involved transduction of a range of hepatic and non-hepatic cell lines as well as primary cells with a virus containing either the CMV immediate early promoter/enhancer driving luciferase [16] or the RSV LTR promoter driving b-galactosidase [17]. High transduction efficiencies and expression levels were observed in primary hepatocytes and hepatoma derived cell lines while low to no expression was seen in other cell types (CHO, HeLa, COS-1, NIH-3T3). Further studies using a recombinant baculovirus carrying the chicken b-actin gene under the control of the CMV immediate early promoter/enhancer showed that non-hepatic cells could also be efficiently transduced [18,19]. Subsequently a large panel of cell lines and primary cultures have been shown to be efficiently transduced with a CMV-GFP baculovirus [20]. High levels of transduction and expression (Fig. 3) were observed in a range of human, rodent and simian cell types as summarised in Table 2. More recent reports have demonstrated the use of recombinant baculovirus for gene expression in human neural [21] and pancreatic islet cells [22]. The ability of this technology to rapidly and efficiently express genes of interest in a variety of commonly used cell types has made it an attractive option for a range of screening assays. Studies have now shown that mammalian cells can be transduced with multiple viruses in a single step, thereby further expanding the potential exploitation of this technology [18,23–25]. In recent years the important role of co-activators (eg., PGC-1, TIF2) and co-repressors (eg., NCoR) in nuclear receptor function has begun to be understood. It is now known that the presence of these cofactors in nuclear receptor reporter assays can have a significant impact on the amplitude and robustness of the response detected. Recombinant baculovirus technology (BacMam), offers a fast and effective way of introducing these cofactors, along with receptor and reporter constructs, into cells in different ratios and combinations. Cell host specific differences in response have also been observed for some nuclear receptors and the large panel of BacMam-transducible cell lines offers the opportunity to study the same receptor–reporter–cofactor combination in multiple cell types.

109 BM-GFP transduction of U-2 OS cells

BM-GFP transduction of HEK293 cells

Fig. 3. Transduction of GFP BacMam virus into U2OS and HEK293 cells. Human U2OS and HEK293 cells were transduced with a BacMam virus carrying the Green Fluorescent Protein gene (GFP) for 72 h. The pictures are at 200 magnification. High efficiency BacMam transduction is clearly visible.

The nuclear receptor superfamily is relatively small consisting of 48 receptors, so in the future BacMam could offer the possibility of efficient nuclear receptor cell based assays on a genome wide scale. Nuclear receptor BacMam virus generation and functional analysis The shuttle plasmid backbone used in the generation of BacMam viruses is simply a modified version of the pFastBac-1 vector (Invitrogen). The polyhedrin promoter used for expression in insect cells was removed and an additional region from pcDNA3 (Invitrogen) was introduced containing the CMV-IE promoter/enhancer for mammalian expression as well as a multiple cloning site and polyadenylation signal. In addition, the SV40 promoter-neomycin phosphotransferase II expression cassette was included for generation of stable cell lines (pFastBacMam-1). The composition of pFastBacMam1 shuttle plasmid is shown in Fig. 4. The inclusion of the pcDNA3 multiple cloning sites helps facilitate the subcloning of nuclear receptor and reporter sequences from other pcDNA3 based vectors into pFastBacMam-1. The process for the generation of BacMam viruses from the pFastBacMam-1 shuttle plasmid is outlined in Fig. 5. This utilises the Bac-to-BacTM (Invitrogen) protocol for production of recombinant baculoviruses. Virus is propagated in Spodoptera frugiperda (Sf9) cells with the addition of serum for stability of the viruses over prolonged periods of time. In the case of nuclear receptors the type of serum used can be critical for the success of downstream assay development.

110 Table 2. Examples of the broad spectrum of mammalian cell types transduced by recombinant baculoviruses. Reprinted from [15] with permission from Elsevier. Human

HeLa Huh7 HEK293 HepG2 KATO-III

Rodent

Non-human Porcine Bovine Ovine Primate

CHO COS7 BHK CV1 RGMI Vero PC12 Mouse Pancreatic b cells IMR32 N2a MT-2 Primary Rat Hepatocytes Pancreatic b cells L929 Keratinocytes Bone Marrow Fibroblasts CHP212 Primary Neural cells W12 SK-N-MC Saos-2 WI38 Primary Hepatocytes FLC4 143TK DLD-1 Embryonic Lung Fibroblasts Primary Foreskin Fibroblasts MRC5 MG63

CPK FS-L3 PK-15

MDB BT

Rabbit

FLL-YFT Primary Hepatocytes

Standard fetal bovine serum (FBS) contains lipids and some steroids that can act as ligands for certain nuclear receptors, which has the potential to produce misleading data. For this reason charcoal stripped/dextran treated serum is added to nuclear receptor BacMams and their corresponding reporter viruses. Once generated, the titre of a BacMam virus can be determined by a number of methods including plaque assays (measuring the number of infectious particles by plaque formation), growth inhibition assays (measuring the inhibition of Sf9 growth) or ELISA techniques (measuring virus particles using an antibody to the gp64 virus cell surface protein). Determination of a viral titre is important in

111

pFastBacMam1 (7433 bp) Tn7L

SV40 polyA

G418 resistance gene

SV40 origin/promoter

BGH polyA

Tn7R

CMV promoter ApaI (4661) XbaI (4655) XhoI (4643) NotI (4636)

Gentamycin resistance

HindIII (4561) KpnI (4567) BamHI (4579) EcoRI (4610)

Fig. 4. The pFastBacMam-1 plasmid backbone used in the generation of the recombinant baculoviruses.

Transformation pFastBac shuttle plasmid

Al

Alkaline lysis

DH10Bac E. coli: site-specific transposition

Add media Transient expression

Transfection Recombinant baculovirus

Sf9 insect cells

Transduction; no viral replication

Virus stock ∼108 pfu/ml

Infection & viral replication

Mammalian cells Sf9 insect cells

Fig. 5. Schematic diagram of BacMam virus generation. Recombinant virus DNA is generated by a site-specific integration process occurring in DH10Bac E. coli cells, and the isolated recombinant viral DNA is then transfected into Sf9 insect cells in which virus replication occurs. The virus can be amplified by infection of fresh insect cell cultures and either purified or used directly to transduce mammalian cells. Reprinted from [15] with permission from Elsevier.

112 the subsequent assay development, where the receptor:reporter ratio can be critical. Transactivation by steroid receptors Development and optimisation of BacMam based nuclear receptor assays is relatively straightforward. A matrix of receptor and reporter (and cofactor) multiplicity of infections (MOIs) can be tested simply by mixing the appropriate volumes of the different viruses with cells and plating in 96-, 384- or 1536-well plates. Cells are incubated in the virus/media mix before compound is added. After further incubation, the plate can be analysed using the appropriate method for reporter read-out. This technique has been successfully utilised for a number of nuclear receptors, both full length receptors and Gal4-LBD fusions. Assessment of BacMam technology for transient expression of full length steroid receptors in mammalian cells was first illustrated in a study of estrogen receptor function in Saos-2 cells [23]. The estrogen receptors a and b recognise the same consensus sequence. Two tandem copies of the estrogen response element (ERE) sequence placed upsteam of a minimal tk promoter were used to replace the CMV promoter expression cassette driving luciferase (Luc) expression to give a pFastBacMam1-ERE-tk-Luc reporter construct. This construct was then used to generate an ERE-tk-Luc BacMam virus. Full length ERa and ERb BacMam viruses were also generated. These viruses have been used in different combinations to transduce T47D cells in the presence or absence of estradiol (E2). After overnight incubation, ER-mediated transcriptional activation could be observed. Luciferase activity was elevated in an E2 dependent manner in both ERa and ERb assays (Fig. 6). Upon treatment with the selective estrogen receptor modulator (SERM) raloxifene, a known ER antagonist in certain tissues, a dosedependent antagonism of this response can also be demonstrated (Fig. 7). In both agonist and antagonist ER BacMam assays the data produced are very similar to those generated in equivalent transient transfection assays, with raloxifene pIC50s varying by only 0.13 nM. The signal to noise windows are also good, making this BacMam system a viable assay format for use in estrogen receptor screening. Other steroid receptors have been studied using BacMam technology. These include the mineralocorticoid receptor (MR) and progesterone receptors A and B (PR-A and PR-B) [24]. These steroid receptors interact with sequences in the mouse mammary tumour virus (MMTV) promoter sequence [26], therefore our studies utilised a single MMTV-Luciferase BacMam as a reporter for these receptors. Evaluation of the MR BacMam assay in CV1 cells indicated a high degree of correlation with respect to potency between BacMam and transient transfection assay formats (Fig. 8). BacMam viruses for PR-A and PR-B have also been generated and validated [24]. The PR-B BacMam was used to investigate the effect of altering the

113

Analysis of ER-alpha & ER-beta Reporter Systems 18000.00 16000.00 14000.00

LCPS

12000.00 10000.00

Mean (N=16) DMSO Mean (N=16) Estradiol

8000.00 6000.00 4000.00 2000.00 0.00 ERE Luciferase

hER-alpha + ERE hER-beta+ ERE Luciferase Luciferase

Fig. 6. Transactivation of ERE-Luc by hERa and hERb in the presence of the ER agonist estradiol. T47D cells were transduced with 200 MOI ERE-Luc BacMam and 50 MOI hERa or hERb BacMam in the presence of estradiol. Clear compound-dependent transactivation was observed with signal to noise windows of 5.29 and 5.21 respectively. LCPS: luciferase counts per second. BacMam

Transient Transfection 110

120

100 90 80

80

%Inhibition

%Inhibition

100

60 40 20

70 60 50 40 30 20

0

10 −20 −12 −11 −10 −9 −8 −7 −6 −5 −4

0 −12 −11 −10 −9 −8 −7 −6

Log Conc (M)

Log Conc (M)

pIC50 = 10.33 nM

pIC50 = 10.20 nM

−5 −4

Fig. 7. Comparison of the cellular potency of raloxifene in an ERa-transient transfection and BacMam antagonist format configured assays. A dose dependent response to the ERa antagonist raloxifene was observed in an assay utilising ERE-Luc and ERa BacMam viruses in T47D cells. This response showed no significant variations from the equivalent transient transfection assay with both producing very similar pIC50 values.

114

9

pEC50 [B]

8

7

6

5

5

5.5

6

6.5

7

7.5

8

8.5

9

9.5

pEC50 [A]

Fig. 8. Correlation coefficient analysis of compounds profiled in the hMR BacMam agonist assay format. A range of compounds were profiled in transient transfection (pEC50[A]) and BacMam (pEC50[B]) assays. Correlation coefficient R2=0.88.

promoter driving receptor expression. The original pFastBacMam vector utilises the CMV promoter. To determine if alternative promoters can be employed, three additional PR-B viruses under the control of the CMV+intron 1, chicken beta actin (CAG) and Elongation Factor 1-alpha (EF1a) promoters were generated. A comparative analysis of the different promoters on the signal to noise window, potency and efficacy was undertaken by profiling a panel of 16 PR agonists. As summarised in Fig. 9, CV1 cells were transduced with a fixed MMTV-luciferase reporter MOI of 100 and varying MOI of the receptors (10, 25 or 50). As had been previously observed using the original PR-B BacMam, altering the multiplicity of infection (MOI) of the receptor did not have an impact on the signal/noise (S/N) window (data not shown). However, comparison of the different promoters demonstrated a rank order profile (EF>CMV-intron ¼ CAG ¼ CMV) in which the EF1a-PR-B virus had a significantly higher S/N window than the original CMV-PR-B virus. This is

115 Analysis of Promoter Variants 4500.00 4000.00 3500.00

LCPS

3000.00

Mean DMSO [N=16]

2500.00 2000.00

Mean 1 µM Progesterone [N=16]

1500.00 1000.00 500.00 0.00 PR-B+MMTV Luciferase

PR-CAG+ PR-CMVint+ MMTV Luciferase MMTV Luciferase

PR-EF+MMTV Luciferase

Fig. 9. The influence of different promoters on the activity of PR-B BacMam. Three PR-B viruses under the control of the CMV+intron 1, chicken beta actin (CAG) and Elongation Factor 1-alpha (EF1a) promoters were generated, transduced into cells together with the MMTV-Luc BacMam and responses to 1 mM progesterone determined.

probably due to the different expression and/or coupling of the four viruses. A multivariate analysis of the potency and efficacy of the four viruses demonstrated very good correlation, particularly with respect to compound potency, indicating that the different expression/coupling efficiencies of these viruses did not alter the pharmacology of the PR-B receptor (Table 3). It remains to be established if the rank order observed with the different promoters holds true for other nuclear receptors. However, these data suggest that BacMam receptors that are poorly expressed or coupled may benefit from the use of different promoters. Moreover, greater expression levels will enable lower viral titres to be used in the BacMam assays thus further enabling the transduction of multiple viruses. The ability to transduce cells with multiple viruses makes possible the addition of cofactors that modulate NR function. PPARg co-activator 1 (PGC-1) is a versatile nuclear receptor co-activator and plays a key role in several pathways including mitochondrial biogenesis, respiration and thermogenesis [27–29]. It is able to interact with several nuclear receptors besides PPARg including GR, PPARa, ER and MR [27,30–32]. This makes PGC-1 a potentially very useful tool in assay development for these targets. The flexibility of the BacMam system has been exploited to investigate the effect of this co-activator on MR activity. Assays were carried out to investigate the effect of adding the PGC-1 BacMam in the MMTV-MR BacMam single shot assay with 100 nM aldosterone (Fig. 10). The addition of PGC-1 BacMam had a clear effect on the signal obtained, increasing the window obtained threefold. Alteration of the MR MOI did not have any effect on the signal window as observed with other steroid receptor BacMams (data not shown). Similar increases in signal in the presence of coactivator have been observed in other NR BacMam assays (including GR and

116 Table 3. Summary of a multivariate analysis of PR-B promoter variants. Correlations (Spreadsheet1) Marked correlations are significant at p<.05000 N=16 (Casewise deletion of missing data) Variable

CMV PR-B

CAG PR-B

CMVint PR-B

EF PR-B

0.90 0.92 1.00 0.78

0.73 0.72 0.78 1.00

0.99 0.99 1.00 0.99

0.98 0.98 0.99 1.00

(A) Multivariate analysis of compound efficacy CMV PR-B CAG PR-B CMVint PR-B EF PR-B

1.00 0.90 0.90 0.73

0.90 1.00 0.92 0.72

(B) Multivariate analysis of compound potency CMV PR-B CAG PR-B CMVint PR-B EF PR-B

1.00 1.00 0.99 0.98

1.00 1.00 0.99 0.98

Analysis of hMR Reporter System 6000.000 Mean (N=16) DMSO 5000.000

Mean (N=16) Aldosterone

LCPS

4000.000

3000.000

2000.000

1000.000

0.000

MMTV Luciferase MMTV Luciferase hMR(100)+ hMR(50)+ hMR(50)+ (200) (200)+PGC-1 MMTV Luciferase MMTV Luciferase MMTV Luciferase (200) (200)+PGC-1 (200) (200)+PGC-1 (200) (200)

Fig. 10. Evaluation of MR-dependent transactivation in the presence of the PGC-1 co-activator BacMam. Addition of PGC-1 BacMam significantly increased the signal in a single 100nM shot MR-MMTV BacMam assay. Variation of MR MOI had no significant effect. Figures in brackets=MOIs.

PPARg; data not shown) indicating the potential for creating highly sensitive assays with this technology. The inclusion of cofactors may also facilitate the generation of cellular models that are more predictive of the biology under investigation.

117

Transrepression by steroid receptors Transpression of NFkB activity by GR is a key mechanism behind the antiinflammatory action of glucocorticoids. NFkB is a transcription factor induced by pro-inflammatory cytokines and plays a crucial role in immunological and inflammatory processes. It regulates transcription of chemoattractants, cytokines such as tumor necrosis factor a (TNFa) and IL-2, cytokine receptors and cell adhesion molecules [33]. Study of this pathway is very important in the development of GR ligands with good anti-inflammatory properties. BacMam technology has been exploited to develop a highly sensitive GR-NFkB transrepression assay with favourable system efficacy. In this assay the activity of a NFkB response element driven luciferase construct is measured in the presence of GR BacMam. The activity of the NFkB-Luc BacMam can be induced by TNFa, and the addition of the GR agonist dexamethasone (Dex) in turn triggers inhibition of NFkB activity resulting in a decrease in the signal observed (Fig. 11A). The transrepression of NFkB activity by GR behaves in a dose-dependent manner with the expected pharmacology (Fig. 11B). The use of BacMam technology has allowed rapid assay development with the selection of the optimal GR:NFkB ratio and host cell line. BacMam technology for assaying PXR transactivation Transactivation of the Pregnane X receptor (PXR) can be used as a tool to assess the developability of compounds for other targets. PXR has been demonstrated to be a key regulator of CYP3A expression in a number of species [34,35]. Cytochrome P450 (CYP) family members play a critical role in the metabolism and detoxification of many endogenous and exogenous compounds to protect against prolonged chemical exposure [36,37]. CYP3A is the predominant isoform of cytochrome P450 in humans which is responsible for the oxidative metabolism of a wide variety of xenobiotics, including an estimated 60% of all clinically used drugs [38]. Drugs that activate CYP3A have the potential to accelerate the metabolism and alter the pharmacological effect of over half of all other therapeutics. Therefore, the importance of CYP3A in drug metabolism means that there is a requirement for routine analysis of PXR activity to assess potential drug liability through its role in regulation of CYP3A induction. Ideally, newly developed drugs should be neutral in their effect on PXR activity and CYP3A expression. Historically, in vitro data on xenobiotic CYP induction has been obtained from primary hepatocytes. Human hepatocytes provide a relevant experimental model but can be difficult to obtain ([39], whereas frequently used rat hepatocytes are easier to obtain but the extrapolation of animal data can be difficult due to marked species differences [40]. Alternatively, other groups have developed cell based hPXR assays [40–42] using a CYP3A promoter with either SPAP or luciferase read out. Although differences in experimental conditions

118 A 16000.00 Mean (N=16) DMSO

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Fig. 11. BacMam viruses can be used in NFkB-GR transrepression assays. (A) NFkB and GR BacMam viruses have been tested in Human Bronchial Smooth Muscle Cells (HBSM), HEK-IP4 and U2OS cells. A clear dexamethasone-dependent transrepression is observed in HEK-IP4 and U2OS cells with very good signals. The data shows averages of 16 assays indicating the robustness of this assay format. (B) NFkB transrepression is dose dependent. Two separate dexamethasone dose–response experiments in HEK-IP4 cells and U2OS cells are shown with the expected pharmacology.

prevent direct comparison of data, the EC50 values for rifampicin range from 200 nM [40] to 8.7 mM [42]. These functional cell-based assays rely on traditional transient transfection of plasmid DNA into host cells and the experiment spans several days. To efficiently assay the PXR activity we have employed BacMam technology. For this assay BacMam viruses for human PXR and a CYP3A4-Luciferase reporter were produced. A direct comparison of hPXR was carried out using BacMam viruses and the pFBM vectors for hPXR and CYP3A4-Luciferase. As shown in Fig. 12, the EC50 values to rifampicin for COS-7 cells transfected with DNA or transduced with BacMam were 1.2 mM and

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Comparison of human PXR activity in COS-7 cells upon DNA transfection or BacMam transduction

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Rifampicin [M] Fig. 12. PXR activity following DNA transfection or BacMam transduction. COS-7 cells were either transfected with hPXR and CYP3A4-Luc DNA constructs or transduced with hPXR and CYP3A4-Luc BacMam viruses. PXR was activated by adding rifampicin and the EC50 of the induction was determined to be EC50 1.2 mM for transient transfection and 730 nM for BacMam transduction.

730 nM respectively. Experimentation with BacMam in further cell lines including HepG2, CV1 and U2OS give EC50 values of 240, 640, and 300 nM respectively (data not shown). The human PXR BacMam provides a robust assay with expected pharmacology in response to a typical tool compound in a range of cell hosts. The BacMam assay can also be carried out in 24 h compared to several days routinely needed for a DNA transient transfection. The adoption of a BacMam based reporter assay for the analysis of PXR in the drug discovery process should result in a quicker turn around time and increased throughput and at the same time reduce assay costs.

The advantages of BacMam technology over existing technologies From its inception the use of recombinant baculovirus technology has been proposed as an alternative to DNA transient transfection or stable cell line generation. As the technology has progressed this has become more and more viable with the BacMam system offering several advantages over the transfectionbased techniques (Table 4).

120 Table 4. Key advantages of the BacMam system. Cost Production time Length of Assay Biosafety profile Cell host range Flexibility Automation Scale-up Cytotoxicity profile Efficiency of gene delivery Ease of matrixing

Significant assay cost reduction due to removal of requirement for expensive lipofection reagents. Virus can be generated from pFastBacMam-1 construct in 2 weeks, much faster than stable cell line generation. Virus can be added at time of cell plating, no prior attachment time required, fewer incubations. Favourable biosafety profile due to replication deficiency in mammalian cells. Ability to transduce a wide range of cell types including some difficult-to-transfect cells. Simple mixing of viruses allows use of multiple viruses in varied ratios within a single experiment. BacMam transduction achieved by simple liquid addition is highly amenable to automation. Simple and economical. Transduction of a large cell population requires only a small volume of virus. Cytotoxicity is significantly reduced compared to transient transfection techniques. High transduction efficiency leads to gene expression in a high percentage of cell population. Multiple viruses can be mixed prior to addition to multiple cell types allowing many combinations to be tested easily.

Stable cell lines have been used for some time in screening strategies. Generation of these involves transfection of cells with the gene of interest and long term culture under a selection agent. The foreign DNA carrying the gene of interest randomly integrates into the host genome in a percentage of transfected cells. Exposure to the selection reagent generates a mass culture of these cells. Pools are then dilution cloned to yield single cell clones which can be scaled-up and tested for expression and function of the gene of interest. Recent advances in automation have streamlined this process somewhat. However, the amount of time and resource required make this process less favoured. Use of stable cell lines also restricts screening to a single cell type. Experience in nuclear receptor assay development has indicated that screening in multiple cell lines is often required to identify the best host for an assay. A quicker alternative to stable cell lines is transient transfection utilising lipid or calcium phosphate precipitation mediated systems for the introduction of foreign plasmid DNA into host cells. In this case, integration of the gene into the host genome is not required. Expression of the gene of interest is transient and transfection must be repeated for each new experiment. The advantage of transient transfection over stable cell line generation is the ability to go directly from plasmid construction to an assay without delay. In addition, multiple DNA constructs can be introduced at the same time in differing amounts making this a much more flexible option. However, this technique also has its drawbacks, most

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critically with regard to the transfection efficiency which impacts the power of transient transfection. The number of cell types that can be used in these assays is limited since certain cell types are difficult to transfect [23]. Variability in transfection efficiency within a single assay system can also be an issue as the percentage of cells successfully transfected can vary between individual experiments. This creates the need for measurement of a second signal (e.g., renilla luciferase as well as firefly luciferase) to normalise the data. Additional issues with transient transfection techniques include the cost of transfection reagents, multi-step protocols and the length of time required for the assays. BacMam technology offers solutions to most of the issues detailed above as well as having other key advantages as summarised in Table 4. The main strength of BacMam over stable cell lines is the relative speed of generation and validation of the viruses. A BacMam virus can be generated in 2–3 weeks from pFastBacMam-1 construct to titred virus with relatively short validation time frames. The turnaround time of individual assays is also reduced from 3 days in historical transient transfections to 24 h with BacMam [24]. This allows higher assay throughput and the ability to run more assays per week. Set up of assays is also accelerated as transduction can be carried out in a single step process as opposed to multiple steps in certain transfection protocols. The straightforward liquid-handling nature of the assay process also makes it highly amenable to automation thereby further streamlining the system and increasing the throughput potential. The scope of individual assays can be expanded using BacMam technology. It is easier to look at multiple variables simply by mixing different volumes of each virus as well as comparing the activity in various cell lines. This offers the potential for multiplexing of different receptors, cofactors and cell lines to provide a fast-track route for assay optimisation. Another important advantage of BacMam technology is its broad cell transduction profile. Green Fluorescent Protein (GFP) BacMam virus can be used to show efficient transduction of a large panel of different cell types including primary cells and transformed cell lines (Fig. 3) [20]. The majority of commonly used cell lines, even those that have low transfection efficiencies, are transduced to levels suitable for compound profiling (Table 2). For example, the percentage of Saos-2 cells expressing GFP after transduction with a GFP BacMam or transfection with pFastBacMam-1GFP DNA was investigated. Almost 100% of transduced cells expressed GFP compared to less than 5% of transfected cells. This group also studied the physical condition of the two treated cultures. Liposome-mediated transfection of Saos-2 cells resulted in significant cytotoxicity which was not observed in the equivalent BacMam transduced culture. These observations along with the positive pharmacological data discussed earlier led these investigators to conclude that BacMam-mediated viral transduction represents a significant improvement over DNA transfection in the estrogen receptor system. Finally, BacMam technology can also offer significant cost-savings over transient transfection methods. Once a BacMam virus has been generated it can

122 be stored in the dark at 4 C for prolonged periods without decrease in titre or activity. Samples from this stock can then be used to quickly amplify large volumes of virus by infection of insect cell cultures. A standard 1 litre batch of virus has been estimated to be sufficient to transduce 200,000 wells or 500384 well plates [24] offering a substantial saving relative to the cost of transfection reagent that would be required. Conclusions One of the key challenges facing the nuclear receptor field is the development of novel compounds which are better at differentiating between the desired and side effect pathways. This NR modulator concept is a sophisticated and complex approach to drug discovery with the aim of developing safer and more selective drugs. This idea is already starting to show its effectiveness with the development of a new generation of ER ligands, selective estrogen receptor modulators (SERMs), which exhibit reduced side effects [43]. This approach is now being adopted by many pharmaceutical and biotechnology companies in the race to develop novel and better therapeutic agents targeting nuclear receptors. The potential for development of new and improved NR modulators has been facilitated by constant advances in the understanding of NR function and the advent of new and exciting technologies in this field. New strategies must now be adopted to allow the drug discovery and development process to effectively assay new leads, reduce cost and decrease cycle time. One of the challenges that has faced the nuclear receptor field has been the development of more sensitive, efficient and relevant assay formats. As discussed in this review, BacMam technology is an ideal alternative for nuclear receptor reporter assays and an excellent tool for high throughput screening. The data presented here demonstrates the utility of BacMam technology in the identification and characterisation of NR ligands. The relative speed, simplicity and the flexibility of this technology will allow expansion of assays to include cofactors or other significant interactors as we learn more about their roles in the function of nuclear receptors. BacMam technology opens new horizons in the exploration of nuclear receptor biology in the quest to discover safer and more effective modulators as therapeutic agents. Acknowledgment The authors wish to thank many of their GSK colleagues for their interest and discussions on BacMam technology. We would like to acknowledge Mike Romanos and John Reardon for their continuous support of BacMam technology within GSK. We would also like to thank Quinn Lu for providing the NFkB-Luc reporter virus.

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