Gene Expression in Mammalian Cells Using BacMam, a Modified Baculovirus System.

BacMams are modified baculoviruses that contain mammalian expression cassettes for gene delivery and expression in mammalian cells. BacMams have become an integral part of the recombinant mammalian gene expression toolbox in research labs worldwide. Construction of transfer vectors is straightforward using basic molecular biology protocols. Virus generation is based on common methods used with the baculovirus insect cell expression system. BacMam transduction of mammalian cells requires minimal modifications to familiar cell culture methods. This chapter highlights the BacMam transfer vector pHTBV.

High throughput screening identifies ATP-competitive inhibitors of the NLRP1 inflammasome.

Nod-like receptors (NLRs) are cytoplasmic pattern recognition receptors that are promising targets for the development of anti-inflammatory therapeutics. Drug discovery efforts targeting NLRs have been hampered by their inherent tendency to form aggregates making protein generation and the development of screening assays very challenging. Herein we report the results of an HTS screen of NLR family member NLRP1 (NLR family, pyrin domain-containing 1) which was achieved through the large scale generation of recombinant GST-His-Thrombin-NLRP1 protein. The screen led to the identification of a diverse set of ATP competitive inhibitors with micromolar potencies. Activity of these hits was confirmed in a FP binding assay, and two homology models were employed to predict the possible binding mode of the leading series and facilitate further lead-optimization. These results highlight a promising strategy for the identification of inhibitors of NLR family members which are rapidly emerging as key drivers of inflammation in human disease.

Identification and characterization of PERK activators by phenotypic screening and their effects on NRF2 activation.

Endoplasmic reticulum stress plays a critical role to restore the homeostasis of protein production in eukaryotic cells. This vital process is hence involved in many types of diseases including COPD. PERK, one branch in the ER stress signaling pathways, has been reported to activate NRF2 signaling pathway, a known protective response to COPD. Based on this scientific rationale, we aimed to identify PERK activators as a mechanism to achieve NRF2 activation. In this report, we describe a phenotypic screening assay to identify PERK activators. This assay measures phosphorylation of GFP-tagged eIF2α upon PERK activation via a cell-based LanthaScreen technology. To obtain a robust assay with sufficient signal to background and low variation, multiple parameters were optimized including GFP-tagged eIF2α BacMam concentration, cell density and serum concentration. The assay was validated by a tool compound, Thapsigargin, which induces phosphorylation of eIF2α. In our assay, this compound showed maximal signal window of approximately 2.5-fold with a pEC50 of 8.0, consistent with literature reports. To identify novel PERK activators through phosphorylation of eIF2α, a focused set of 8,400 compounds was screened in this assay at 10 µM. A number of hits were identified and validated. The molecular mechanisms for several selected hits were further characterized in terms of PERK activation and effects on PERK downstream components. Specificity of these compounds in activating PERK was demonstrated with a PERK specific inhibitor and in PERK knockout mouse embryonic fibroblast (MEF) cells. In addition, these hits showed NRF2-dependent anti-oxidant gene induction. In summary, our phenotypic screening assay is demonstrated to be able to identify PERK specific activators. The identified PERK activators could potentially be used as chemical probes to further investigate this pathway as well as the link between PERK activation and NRF2 pathway activation.

Optimized procedures for producing biologically active chemokines.

We describe here two strategies to produce biologically active chemokines with authentic N-terminal amino acid residues. The first involves producing the target chemokine with an N-terminal 6xHis-SUMO tag in Escherichia coli as inclusion bodies. The fusion protein is solubilized and purified with Ni-NTA-agarose in denaturing reagents. This is further followed by tag removal and refolding in a redox refolding buffer. The second approach involves expressing the target chemokine with an N-terminal 6xHis-Trx-SUMO tag in an engineered E. coli strain that facilitates formation of disulfide bonds in the cytoplasm. Following purification of the fusion protein via Ni-NTA and tag removal, the target chemokine is refolded without redox buffer and purified by reverse phase chromatography. Using the procedures, we have produced more than 15 biologically active chemokines, with a yield of up to 15 mg/L.

Gene expression in mammalian cells using BacMam, a modified baculovirus system.

BacMams are modified baculoviruses that contain mammalian expression cassettes for gene delivery and expression in mammalian cells. The BacMam system combines the advantages of viral transient expression, ease in generation, and a wide cell tropism. It enables rapid, facile, and flexible gene over-expression experiments to be performed in a variety of mammalian cell lines. Conversion of baculovirus vectors to BacMam vectors involves replacement of the viral specific expression cassette with a mammalian expression cassette or the addition of a mammalian expression cassette. Viruses are produced using standard methods in a few weeks. Mammalian cells transduced with the BacMam viruses have been routinely used as substitutes for stable cell lines.

Oxazolidinones as novel human CCR8 antagonists.

High-throughput screening of the corporate compound collection led to the discovery of a novel series of N-substituted-5-aryl-oxazolidinones as potent human CCR8 antagonists. The synthesis, structure-activity relationships, and optimization of the series that led to the identification of SB-649701 (1a), are described.

Pharmacological regulation of insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identification of agonist and antagonist small molecules.

1. Long chain fatty acids have recently been identified as agonists for the G protein-coupled receptors GPR40 and GPR120. Here, we present the first description of GW9508, a small-molecule agonist of the fatty acid receptors GPR40 and GPR120. In addition, we also describe the pharmacology of GW1100, a selective GPR40 antagonist. These molecules were used to further investigate the role of GPR40 in glucose-stimulated insulin secretion in the MIN6 mouse pancreatic beta-cell line. 2. GW9508 and linoleic acid both stimulated intracellular Ca2+ mobilization in human embryonic kidney (HEK)293 cells expressing GPR40 (pEC50 values of 7.32+/-0.03 and 5.65+/-0.06, respectively) or GPR120 (pEC50 values of 5.46+/-0.09 and 5.89+/-0.04, respectively), but not in the parent HEK-293 cell line. 3. GW1100 dose dependently inhibited GPR40-mediated Ca2+ elevations stimulated by GW9508 and linoleic acid (pIC50 values of 5.99+/-0.03 and 5.99+/-0.06, respectively). GW1100 had no effect on the GPR120-mediated stimulation of intracellular Ca2+ release produced by either GW9508 or linoleic acid. 4. GW9508 dose dependently potentiated glucose-stimulated insulin secretion in MIN6 cells, but not in primary rat or mouse islets. Furthermore, GW9508 was able to potentiate the KCl-mediated increase in insulin secretion in MIN6 cells. The effects of GW9508 on insulin secretion were reversed by GW1100, while linoleic acid-stimulated insulin secretion was partially attenuated by GW1100. 5. These results add further evidence to a link between GPR40 and the ability of fatty acids to acutely potentiate insulin secretion and demonstrate that small-molecule GPR40 agonists are glucose-sensitive insulin secretagogues.

The peptidic urotensin-II receptor ligand GSK248451 possesses less intrinsic activity than the low-efficacy partial agonists SB-710411 and urantide in native mammalian tissues and recombinant cell systems.

Several peptidic urotensin-II (UT) receptor antagonists exert 'paradoxical' agonist activity in recombinant cell- and tissue-based bioassay systems, likely the result of differential urotensin-II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT-HEK (human embryonic kidney) cells.BacMam-mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U-II>urantide ([Pen(5)-DTrp(7)-Orn(8)]hU-II(4-11))>SB-710411 (Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-amide)>>GSK248451 (Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-amide) (the relative coupling efficiency of recombinant HEK cells was cat>human>>rat UT receptor). The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low-efficacy agonists appear to function as antagonists. In contrast to the 'low-efficacy agonists' urantide and SB-710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4-5-fold less than seen with urantide). Since GSK248451 (1 mg kg(-1), i.v.) blocked the systemic pressor actions of exogenous U-II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U-II in the aetiology of mammalian cardiometabolic diseases.

BacMam recombinant baculoviruses in G protein-coupled receptor drug discovery.

With completion of the sequencing of the human and mouse genomes, the primary sequences of close to 400 non-olfactory G protein-coupled receptors (GPCRs) have been determined. There are intensive efforts within the pharmaceutical industry to discover and develop new therapeutic agents acting via GPCRs. In addition, there is a concerted effort to identify potential new drug targets from the remaining 150+orphan GPCRs through the identification of their ligands. Access to functionally expressed recombinant receptors underpins both of these key drug discovery activities. Typically, GPCR drug discovery screening activities are carried out using mammalian cell lines stably expressing the target of interest. The influx of new receptor sequences originating from genomic sequencing efforts has caused a shift toward wider applications of transient rather than stable expression systems, especially in support of assays for orphan receptor ligand screening. Recombinant baculoviruses in which the polyhedrin promoter has been replaced with a mammalian promoter, termed BacMam viruses, were originally designed as potential new gene therapy delivery vehicles. This same technology offers numerous advantages as a transient expression system in the assay of membrane-expressed drug targets, including GPCRs. Data presented show that BacMam can be used rapidly to generate robust and pharmacologically authentic GPCR assays in several formats, with the potential to transform drug discovery screening processes for this gene family.

Identification of novel inhibitors of the transforming growth factor beta1 (TGF-beta1) type 1 receptor (ALK5).

Screening of our internal compound collection for inhibitors of the transforming growth factor beta1 (TGF-beta1) type I receptor (ALK5) identified several hits. Optimization of the dihydropyrroloimidazole hit 2 by introduction of a 2-pyridine and 3,4-methylenedioxyphenyl group gave 7, a selective ALK5 inhibitor. With this information, optimization of the triarylimidazole hit 8 gave the selective inhibitor 14, which inhibits TGF-beta1-induced fibronectin mRNA formation while displaying no measurable cytotoxicity in the 48 h XTT assay.

Rapid expression of recombinant proteins in modified CHO cells using the baculovirus system.

Baculovirus containing the mammalianCMV promoter, in place of the insect polyhedronpromoter (BacMam), has been used to transientlytransfect COS, CHO and CHOE1a (CHO cells expressing theE1a transcriptional activator). Using this system forthe expression of a cellular adhesion factor (SAF-3) Fcfusion protein in CHOE1a, we found that levels ofexpression were highest with a MOI of 100, 20mM sodiumbutyrate, at 34 degrees C. Production increased furtherif the cells were resuspended in fresh medium, about3 x 10(6) cells ml(-1), prior to addition of the virus. These conditions were used to express 3 secretedproteins, SAF-3-Fc, CD40-hexa his and Asp 2-Fc, and, at2 to 6 days post infection, protein levels ranged from4 ug ml(-1) to 25 ug ml(-1). Based on these results, theBacMam system represents a viable technique forproducing protein at ug ml(-1) levels in a relatively shortperiod of time.