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Biomedical subjects

Brian A Pollok

Publications and source records attributed to Brian A Pollok.

6 recordsLinked to original sources
PubMed2005

Diverse small-molecule modulators of SMN expression found by high-throughput compound screening: early leads towards a therapeutic for spinal muscular atrophy.

We have exploited the existence of a second copy of the human SMN gene (SMN2) to develop a high-throughput screening strategy to identify potential small molecule therapeutics for the genetic disease spinal muscular atrophy (SMA), which is caused by the loss of the SMN1 gene. Our screening process was designed to identify synthetic compounds that increase the total amount of full-length SMN messenger RNA and protein arising from the SMN2 gene, thereby suppressing the deleterious effects of losing SMN1. A cell-based bioassay was generated that detects SMN2 promoter activity, on which greater than 550,000 compounds was tested. This resulted in the identification of 17 distinct compounds with confirmed biological activity on the cellular primary assay, belonging to nine different structural families. Six of the nine scaffolds were chosen on the basis of their drug-like features to be tested for their ability to modulate SMN gene expression in SMA patient-derived fibroblasts. Five of the six compound classes altered SMN mRNA levels or mRNA splicing patterns in SMA patient-derived fibroblasts. Two of the compound classes, a quinazoline compound series and an indole compound, also increased SMN protein levels and nuclear gem/Cajal body numbers in patient-derived cells. In addition, these two distinct scaffolds showed additive effects when used in combination, suggesting that they may act on different molecular targets. The work described here has provided the foundation for a successful medicinal chemistry effort to further advance these compounds as potential small molecule therapeutics for SMA.

Base Sequence↗
PubMed2004

Sortase-mediated protein ligation: a new method for protein engineering.

Sortase (SrtA), a transpeptidase from Staphylococcus aureus, catalyzes a cell-wall sorting reaction at an LPXTG motif by cleaving between threonine and glycine and subsequently joining the carboxyl group of threonine to an amino group of pentaglycine on the cell wall peptidoglycan. We have applied this transpeptidyl activity of sortase to in vitro protein ligation. We found that in the presence of sortase, protein/peptide with an LPXTG motif can be specifically ligated to an aminoglycine protein/peptide via an amide bond. Additionally, sortase can even conjugate substrates such as (d)-peptides, synthetic branched peptides, and aminoglycine-derivatized small molecules to the C terminus of a recombinant protein. The sortase-mediate protein ligation is robust, specific, and easy to perform, and can be widely applied to specific protein conjugation with polypeptides or molecules of unique biochemical and biophysical properties.

Amino Acid Sequence↗
PubMed2003

Gain-of-function somatic cell lines for drug discovery applications generated by homologous recombination.

Gene targeting allows for precise genomic engineering and has been used extensively to generate both loss-of-function and gain-of-function models in mice. Similar manipulation of the genome of somatic cell lines holds high value in basic and applied research, but has been hampered by low recombination frequencies and the subsequent labor-intensive analysis of a large number of cell clones. By combining gene targeting methods with fluorescence-activated cell sorting, gain-of-function cell lines were generated and identified based on a functional readout. To demonstrate the general applicability of this approach to drug discovery, we generated targeted promoter insertion cell lines for two key drug target classes -- the G protein-coupled receptor melanocortin-receptor 4 and the nuclear receptor peroxisome proliferator-activated receptor-gamma. Molecular analysis of the engineered cell clones confirmed the predicted integration of a constitutive promoter into an endogenous allele, and the appropriate pharmacology for these targets validated the use of these gain-of-function cell lines in drug discovery applications, including high-throughput compound screening.

Cell Line↗
PubMed2002

A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases.

Protein phosphorylation is one of the major regulatory mechanisms involved in signal-induced cellular events, including cell proliferation, apoptosis, and metabolism. Because many facets of biology are regulated by protein phosphorylation, aberrant kinase and/or phosphatase activity forms the basis for many different types of pathology. The disease relevance of protein kinases and phosphatases has led many pharmaceutical and biotechnology companies to expend significant resources in lead discovery programs for these two target classes. The existence of >500 kinases and phosphatases encoded by the human genome necessitates development of methodologies for the rapid screening for novel and specific compound inhibitors. We describe here a fluorescence-based, molecular assay platform that is compatible with robotic, ultra-high throughput screening systems and can be applied to virtually all tyrosine and serine/threonine protein kinases and phosphatases. The assay has a coupled-enzyme format, utilizing the differential protease sensitivity of phosphorylated versus nonphosphorylated peptide substrates. In addition to screening individual kinases, the assay can be formatted such that kinase pathways are re-created in vitro to identify compounds that specifically interact with inactive kinases. Miniaturization of this assay format to the 1-microl scale allows for the rapid and accurate compound screening of a host of kinase and phosphatase targets, thereby facilitating the hunt for new leads for these target classes.

Adenosine Triphosphate↗
PubMed2002

Binding of a Pleckstrin homology domain protein to phosphoinositide in membranes: a miniaturized FRET-based assay for drug screening.

Pleckstrin homology (PH) domains are present in key proteins involved in many vital cell processes. For example, the PH domain of Bruton's tyrosine kinase (Btk) binds to phosphatidylinositol triphosphate (PIP(3)) in the plasma membrane after stimulation of the B-cell receptor in B cells. Mutations in the Btk PH domain result in changes in its affinity for PIP(3), with higher binding leading to cell transformation in vitro and lower binding leading to antibody deficiencies in both humans and mice. We describe here a fluorescence resonance energy transfer (FRET)-based biochemical assay that directly monitors the interaction of a PH domain with PIP(3) at a membrane surface. We overexpressed a fusion protein consisting of an enhanced green fluorescent protein (GFP) and the N-terminal 170 amino acids of a Tec family kinase that contains its PH domain (PH170). Homogeneous unilamellar vesicles were made that contained PIP(3) and octadecylrhodamine (OR), a lipophilic FRET acceptor for GFP. After optimization of both protein and vesicle components, we found that binding of the GFP-PH170 protein to PIP3 in vesicles that contain OR results in about a 90% reduction of GFP fluorescence. Using this assay to screen 1440 compounds, we identified three that efficiently inhibited binding of GFP-PH170 to PIP(3) in vesicles. This biochemical assay readily miniaturized to 1.8-microl reaction volumes and was validated in a 3456-well screening format.

Agammaglobulinaemia Tyrosine Kinase↗