Development and validation of an improved inducer-regulator protein complex in the pBRES-regulated expression system.

Widespread adaptation of small molecule-regulated expression systems requires the development of selective inducer molecules that do not have any significant side effects on the endogenous receptors from which the regulated expression system is derived. Here we report the identification and in vitro validation of a novel inducer-receptor pair for the single-plasmid regulated expression system termed pBRES, which contains the ligand-binding domain from the human progesterone receptor (hPR). A small molecule inducer, BLX-913, has been identified as having a 30-fold lower IC(50) for the human progesterone receptor than mifepristone (MFP), the previously best characterized inducer for pBRES. Using modeling-guided protein engineering, compensatory mutations were installed at positions W755 and V729 (hPR numbering) in the ligand-binding pocket of the pBRES regulator protein (pBRES RP) to accommodate the new inducer and allow induction of transgene expression to levels previously seen with MFP. The improved inducer-pBRES RP complex was validated in vitro by monitoring the induction of luciferase, murine secreted alkaline phosphatase, and human interferon beta transgenes in mouse skeletal muscle cells. The engineered pBRES demonstrated low levels of transgene expression in the absence, and high expression levels in the presence, of the new BLX-913 inducer. Findings presented here allow induction of the pBRES-regulated gene expression system by a compound with markedly lower anti-hPR activity than MFP, the previously best characterized inducer.

A novel high-throughput screening format to identify inhibitors of secreted acid sphingomyelinase.

Secreted extracellular acid sphingomyelinase (sASM) activity has been suggested to promote atherosclerosis by enhancing subendothelial aggregation and retention of low-density lipoprotein (LDL) with resultant foam cell formation. Compounds that inhibit sASM activity, at neutral pH, may prevent lipid retention and thus would be expected to be anti-atherosclerotic. With the goal of identifying novel compounds that inhibit sASM at pH 7.4, a high-throughput screen was performed. Initial screening was run using a modification of a proven system that measures the hydrolysis of radiolabeled sphingomyelin presented in detergent micelles in a 96-well format. Separation of the radiolabeled aqueous phosphorylcholine reaction product from uncleaved sphingomyelin lipid substrate was achieved by chloroform/methanol extraction. During the screening campaign, a novel extraction procedure was developed to eliminate the use of the hazardous organic reagents. This new procedure exploited the ability of uncleaved, radiolabeled lipid substrate to interact with hydrophobic phenyl-sepharose beads. A comparison of the organic-based and the bead-based extraction sASM screening assays revealed Z' factor values ranging from 0.7 to 0.95 for both formats. In addition, both assay formats led to the identification of sub- to low micromolar inhibitors of sASM at pH 7.4 with similar IC(50) values. Subsequent studies demonstrated that both methods were also adaptable to run in a 384-well format. In contrast to the results observed at neutral pH, however, only the organic extraction assay was capable of accurately measuring sASM activity at its pH optimum of 5.0. The advantages and disadvantages of both sASM assay formats are discussed.

A model of the acid sphingomyelinase phosphoesterase domain based on its remote structural homolog purple acid phosphatase.

Sequence profile and fold recognition methods identified mammalian purple acid phosphatase (PAP), a member of a dimetal-containing phosphoesterase (DMP) family, as a remote homolog of human acid sphingomyelinase (ASM). A model of the phosphoesterase domain of ASM was built based on its predicted secondary structure and the metal-coordinating residues of PAP. Due to the low sequence identity between ASM and PAP (approximately 15%), the highest degree of confidence in the model resides in the metal-binding motifs. The ASM model predicts residues Asp 206, Asp 278, Asn 318, His 425, and His 457 to be dimetal coordinating. A putative orientation for the phosphorylcholine head group of the ASM substrate, sphingomyelin (SM), was made based on the predicted catalysis of the phosphorus-oxygen bond in the active site of ASM and on a structural comparison of the PAP-phosphate complex to the C-reactive protein-phosphorylcholine complex. These complexes revealed similar spatial interactions between the metal-coordinating residues, the metals, and the phosphate groups, suggesting a putative orientation for the head group in ASM consistent with the mechanism considerations. A conserved sequence motif in ASM, NX3CX3N, was identified (Asn 381 to Asn 389) and is predicted to interact with the choline amine moiety in SM. The resulting ASM model suggests that the enzyme uses an SN2-type catalytic mechanism to hydrolyze SM, similar to other DMPs. His 319 in ASM is predicted to protonate the ceramide-leaving group in the catalysis of SM. The putative functional roles of several ASM Niemann-Pick missense mutations, located in the predicted phosphoesterase domain, are discussed in context to the model.