Fragment Binding to ß-Secretase 1 without Catalytic Aspartate Interactions Identified via Orthogonal Screening Approaches.

An approach to identify ß-secretase 1 (BACE1) fragment binders that do not interact with the catalytic aspartate dyad is presented. A ThermoFluor (thermal shift) and a fluorescence resonance energy transfer enzymatic screen on the soluble domain of BACE1, together with a surface plasmon resonance (SPR) screen on the soluble domain of BACE1 and a mutant of one catalytic Asp (D32N), were run in parallel. Fragments that were active in at least two of these assays were further confirmed using one-dimensional NMR (WaterLOGSY) and SPR binding competition studies with peptidic inhibitor OM99-2. Protein-observed NMR (two-dimensional 15N heteronuclear single-quantum coherence spectroscopy) and crystallographic studies with the soluble domain of BACE1 identified a unique and novel binding mode for compound 12, a fragment that still occupies the active site while not making any interactions with catalytic Asps. This novel approach of combining orthogonal fragment screening techniques, for both wild-type and mutant enzymes, as well as binding competition studies could be generalized to other targets to overcome undesired interaction motifs and as a hit-generation approach in highly constrained intellectual property space.

Cell-Free Expression of G Protein-Coupled Receptors.

The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes.

Recombinant expression, purification and dimerization of the neurotrophic growth factor Artemin for in vitro and in vivo use.

Artemin (ARTN) is a neurotrophic growth factor of the GDNF ligand family that signals through the specific GFRα-3 coreceptor/cRet tyrosine kinase-mediated signaling cascade. Its expression and signaling action in adults are restricted to nociceptive sensory neurons in the dorsal root ganglia. Consequently, Artemin supports survival and growth of sensory neurons and has been studied as a possible treatment for neuropathic pain paradigms. In this paper, we describe the development of an efficient method for the recombinant bacterial production of large quantities of highly pure, biologically active ARTN for in vitro and in vivo studies. Using Escherichia coli expression of an NH(2)-terminal SUMO-Artemin fusion protein and subsequent refolding from inclusion bodies followed by cleavage of the SUMO fusion part, mature Artemin with a native NH(2)-terminal amino acid sequence was obtained at high purity (>99%). Experiments using the reducing agent dithiothreitol (DTT) demonstrated that the intermolecular disulphide bridge in the cysteine knot is dispensable for dimerization of stable ARTN monomers. Our production method could facilitate in vitro and in vivo experimentation for the possible development of Artemin as a therapeutic agent for neuropathic pain.

Exceptional stability of artemin neurotrophic factor dimers: effects of temperature, pH, buffer and storage conditions on protein integrity and activity.

Artemin (ARTN) is a neurotrophic growth factor of the GDNF ligand family that signals through the specific GFRα-3 coreceptor/cRet tyrosine kinase-mediated signaling cascade. Its expression and signaling action in adults are restricted to nociceptive sensory neurons in the dorsal root ganglia. Consequently, Artemin supports survival and growth of sensory neurons and has been studied as a possible treatment for neuropathic pain. We have developed a robust and sensitive cellular assay to measure ARTN biological activity. Using recombinant Artemin produced in Escherichia coli bacteria together with this specific assay, we demonstrate that ARTN is an exceptionally stable polypeptide. Multiple freeze-thaw cycles, incubation at elevated temperatures (up to 90 °C) for 0.5 h, prolonged storage at 4 °C, and exposure to conditions of different pH, salt concentration, and additives had no measurable effect on the biological activity of ARTN. In some of the tested conditions, partial removal of nine NH(2)-terminal amino acids of the ARTN protein occurred, but this truncation had no important effect on the ARTN signaling response. Consequently, we postulate that formulation and storage for in vivo testing of ARTN in neuropathic pain paradigms in animals and humans should be straightforward.

2-Amino-3,4-dihydroquinazolines as inhibitors of BACE-1 (beta-site APP cleaving enzyme): Use of structure based design to convert a micromolar hit into a nanomolar lead.

A new aspartic protease inhibitory chemotype bearing a 2-amino-3,4-dihydroquinazoline ring was identified by high-throughput screening for the inhibition of BACE-1. X-ray crystallography revealed that the exocyclic amino group participated in a hydrogen bonding array with the two catalytic aspartic acids of BACE-1 (Asp(32), Asp(228)). BACE-1 inhibitory potency was increased (0.9 microM to 11 nM K(i)) by substitution into the unoccupied S(1)' pocket.

Recombinant insect cell expression and purification of human beta-secretase (BACE-1) for X-ray crystallography.

Human beta-secretase (BACE-1) is a type I integral membrane aspartic protease that catalyzes the internal cleavage of the amyloid precursor protein (APP), generating the N-terminus of the Abeta peptide. The generation and subsequent extracellular deposition of Abeta(1-42) peptide into amyloid plaques in the brain constitute one of the hallmarks of Alzheimer's disease (AD), a common debilitating neurodegenerative disorder. Inhibition of BACE-1 is considered an excellent therapeutic strategy against AD. To generate pure enzyme for protein crystallography and subsequent structure-based drug design, we have expressed a soluble, unglycosylated, 6xHis-tagged form of proBACE-1 in insect cells using baculovirus infection. To avoid production of a mixture of the pro-enzyme form and the mature form of BACE-1, the proprotein convertase furin was coexpressed with proBACE-1, leading to almost complete proteolytic activation of the recombinant enzyme. The mature enzyme was secreted in the conditioned medium of BACE-1/furin coinfected HighFive insect cells. Secreted BACE-1 protein was purified to homogeneity from the medium using subsequent Ni-chelate affinity chromatography, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. To avoid autoproteolysis, all purification steps were performed at pH values outside the activity range of BACE-1. The purified, biologically active enzyme was homogeneous on SDS/PAGE and had the expected sequence and molecular mass determined by N-terminal amino acid sequencing and mass spectrometry, respectively. Moreover, the preparation showed a single peak of the expected size with only 17% polydispersity using dynamic light scattering analysis. The yield of BACE-1 from fermentation cultures was approximately 0.1mg pure enzyme per liter of cell culture medium. The purified protein was successfully used to generate BACE-1/inhibitor co-crystals and to determine the crystal structure of the complex by X-ray analysis. The availability of substantial quantities of active, homogeneous enzyme will be of great help in future structure-based drug design efforts in the search for efficient protease inhibitor drugs to treat AD.

Characterization of platelet-derived growth factor-C (PDGF-C): expression in normal and tumor cells, biological activity and chromosomal localization.

The predicted platelet-derived growth factor-C (PDGF-C) polypeptide contains an N-terminal CUB-like domain and a C-terminal domain with homology to members of the PDGF/vascular endothelial growth factor (VEGF) family. PDGF-C mRNA is widely expressed in normal tissues and does not appear to be up-regulated in the tumor cell lines tested. The PDGF-C gene was mapped to human chromosome 4q31-32. PDGF-C protein and the CUB domain of PDGF-C expressed in Escherichia coli, were able to stimulate proliferation of human artery smooth muscle cells, but were inactive on umbilical vein endothelial cells, osteoblasts, fibroblasts, skeletal muscle cells (SkMC), bovine chondrocytes, and rat myocardium cells. Although the mitogenic activity of PDGF-C and the CUB domain was only observed at concentrations ranging from 1 to 10 microg/ml, substitution of Cys(124) by Ser or deletion of Cys(124) significantly reduced the mitogenic activity. Our data suggest a possible role of the CUB domain of PDGF-C in addition to its role in maintaining latency of the PDGF domain.