Development of a fluorescence polarization binding assay for folate receptor.

Folate receptor (FR) has been actively investigated for targeted delivery of therapeutics into cancer cells because this receptor is selectively and highly expressed in carcinomas. Because FR rapidly cycles between the cell surface and cytoplasm, folic acid conjugated to a therapeutic agent can drive targeted therapeutic delivery to cancer cells. We prepared a novel fluorescent ligand Cy5-folate and used it to develop a fluorescence polarization (FP) FR binding assay to determine the binding affinities of FR-targeted molecules. The assay was performed in 96-well microplates using membrane preparations from human KB cells as a source of FR and Cy5 fluorophore-labeled folic acid as a tracer. This high-throughput homogeneous assay demonstrates advantages over existing multistep methods in that it minimizes both time and resources spent determining binding affinities. At the optimized conditions, a Z' of 0.64 was achieved in a 96-well format.

Development of a fluorescence polarization binding assay for asialoglycoprotein receptor.

Asialoglycoprotein receptor (ASGP-R) has been actively investigated for targeted delivery of therapeutic agents into hepatocytes because this receptor is selectively and highly expressed in liver and has a high internalization rate. Synthetic cluster glycopeptides (e.g., triGalNAc) bind with high affinity to ASGP-R and, when conjugated to a therapeutic agent, can drive receptor-mediated uptake in liver. We developed a novel fluorescent polarization (FP) ASGP-R binding assay to determine the binding affinities of ASGP-R-targeted molecules. The assay was performed in 96-well microplates using membrane preparations from rat liver as a source of ASGP-R and Cy5 fluorophore-labeled triGalNAc synthetic ligand as a tracer. This high-throughput homogeneous assay demonstrates advantages over existing multistep methods in that it minimizes both time and resources spent in determining binding affinities to ASGP-R. At the optimized conditions, a Z' factor of 0.73 was achieved in a 96-well format.

Development of a microplate fluorescence assay for kynurenine aminotransferase.

Inhibition of kynurenine aminotransferases (KATs) is a strategy to therapeutically reduce levels of kynurenic acid (KYNA), an endogenous antagonist of glutamatergic N-methyl-D-aspartate (NMDA) and cholinergic α7 nicotinic receptors. Several methods of measuring KAT activity in vitro have been developed, but none is well-suited to high throughput and automation. In this article, we describe a modification of existing high-performance liquid chromatography (HPLC)-based methods that enables the development of a 96-well microplate assay in both enzyme- and cell-based formats using human KAT I as an example. KYNA enzymatically produced from L-kynurenine is measured directly in a reaction mixture fluorimetrically.

Deducing the transmembrane domain organization of presenilin-1 in gamma-secretase by cysteine disulfide cross-linking.

Gamma-secretase is a founding member of membrane-embedded aspartyl proteases that cleave substrates within transmembrane domains, and this enzyme is an important target for the development of therapeutics for Alzheimer's disease. The structure of gamma-secretase and its precise catalytic mechanism still remain largely unknown. Gamma-secretase is a complex of four integral membrane proteins, with presenilin (PS) as the catalytic component. To gain structural and functional information about the nine-transmembrane domain (TMD) presenilin, we employed a cysteine mutagenesis/disulfide cross-linking approach. Here we report that native Cys92 is close to both Cys410 and Cys419, strongly implying that TMD1 and TMD8 are adjacent to each other. This structural arrangement also suggests that TMD8 is distorted from an ideal helix. Importantly, binding of an active site directed inhibitor, but not a docking site directed inhibitor, reduces the ability of the native cysteine pairs of PS1 to cross-link upon oxidation. These findings suggest that the conserved cysteines of TMD1 and TMD8 contribute to or allosterically interact with the active site of gamma-secretase.

The initial substrate-binding site of gamma-secretase is located on presenilin near the active site.

gamma-Secretase is a structurally enigmatic multiprotein complex that catalyzes intramembrane proteolysis of a variety of substrates, including the amyloid beta-protein precursor of Alzheimer's disease and the Notch receptor essential to cell differentiation. The active site of this transmembrane aspartyl protease apparently lies at the interface between two subunits of presenilin-1 (PS1); however, evidence suggests the existence of an initial substrate-binding site that is distinct from the active site. Here, we report that photoaffinity probes based on potent helical peptide inhibitors and designed to mimic the amyloid beta-protein precursor substrate bind specifically to the PS subunit interface, at a site close to the active site. The location of the helical peptide-binding site suggests that substrate passes between the two PS1 subunits to access the active site. An aggressive Alzheimer-causing mutation in PS1 strongly reduced photolabeling by a transition-state analogue but not by helical peptides, providing biochemical evidence that the pathological effect of this PS mutation is due to alteration of the active-site topography.

Signal peptide peptidase forms a homodimer that is labeled by an active site-directed gamma-secretase inhibitor.

Presenilin (PS) is the presumptive catalytic component of the intramembrane aspartyl protease gamma-secretase complex. Recently a family of presenilin homologs was identified. One member of this family, signal peptide peptidase (SPP), has been shown to be a protease, which supports the hypothesis that PS and presenilin homologs are related intramembrane-cleaving aspartyl proteases. SPP has been reported as a glycoprotein of approximately 45 kDa. Our initial characterization of SPP isolated from human brain and cell lines demonstrated that SPP is primarily present as an SDS-stable approximately 95-kDa protein on Western blots. Upon heating or treatment of this approximately 95-kDa SPP band with acid, a approximately 45-kDa band could be resolved. Co-purification of two different epitope-tagged forms of SPP from a stably transfected cell line expressing both tagged versions demonstrated that the approximately 95-kDa band is a homodimer of SPP. Pulse-chase metabolic labeling studies demonstrated that the SPP homodimer assembles rapidly and is metabolically stable. In a glycerol velocity gradient, SPP sedimented from approximately 100-200 kDa. Significantly the SPP homodimer was specifically labeled by an active site-directed photoaffinity probe (III-63) for PS, indicating that the active sites of SPP and PS/gamma-secretase are similar and providing strong evidence that the homodimer is functionally active. Collectively these data suggest that SPP exists in vivo as a functional dimer.

Differential effects of inhibitors on the gamma-secretase complex. Mechanistic implications.

Gamma-secretase is a protease complex of four integral membrane proteins, with presenilin (PS) as the apparent catalytic component, and this enzyme processes the transmembrane domains of a variety of substrates, including the amyloid beta-protein precursor and the Notch receptor. Here we explore the mechanisms of structurally diverse gamma-secretase inhibitors by examining their ability to displace an active site-directed photoprobe from PS heterodimers. Most gamma-secretase inhibitors, including a potent inhibitor of the PS-like signal peptide peptidase, blocked the photoprobe from binding to PS1, indicating that these compounds either bind directly to the active site or alter it through an allosteric interaction. Conversely, some reported inhibitors failed to displace this interaction, demonstrating that these compounds do not interfere with the protease by affecting its active site. Differential effects of the inhibitors with respect to photoprobe displacement and in cell-based and cell-free assays suggest that these compounds are important mechanistic tools for deciphering the workings of this intramembrane-cleaving protease complex and its similarity to other polytopic aspartyl proteases.