A Solid Supported Membrane-Based Technology for Electrophysical Screening of BAT1-Modulating Compounds.

Classical high-throughput screening (HTS) technologies for the analysis of ionic currents across biological membranes can be performed using fluorescence-based, radioactive, and mass spectrometry (MS)-based uptake assays. These assays provide rapid results for pharmacological HTS, but the underlying, indirect analytical character of these assays can be linked to high false-positive hit rates. Thus, orthogonal and secondary assays using more biological target-based technologies are indispensable for further compound validation and optimization.

Matching pH values for antibody stabilization and crystallization suggest rationale for accelerated development of biotherapeutic drugs.

Monoclonal antibodies (mAbs) are currently leading products in the global biopharmaceutical market. Multiple mAbs are in clinical development and novel biotherapeutic protein scaffolds, based on the canonical immunoglobulin G (IgG) fold, are emerging as treatment options for various medical conditions. However, fast approvals for biotherapeutics are challenging to achieve, because of difficult scientific development procedures and complex regulatory processes.

The Keap1-Nrf2 protein-protein interaction: A suitable target for small molecules.

The transcription factor Nrf2 controls pathways involved in oxidative-stress defense and is a potential pharmacological target for the treatment of chronic diseases. Activators of Nrf2 that have undergone clinical development are reactive molecules that are either associated with safety issues or for which it is unclear if their pharmacological efficacy depends on the activation of Nrf2. Therefore, the clinical validity of Nrf2 activation is not yet proven.

Characterization of RA839, a Noncovalent Small Molecule Binder to Keap1 and Selective Activator of Nrf2 Signaling.

The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of ∼6 μM, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry.

1,1-Dioxo-5,6-dihydro-[4,1,2]oxathiazines, a novel class of 11ß-HSD1 inhibitors for the treatment of diabetes.

Racemic cis-1,1-dioxo-5,6-dihydro-[4,1,2]oxathiazine derivative 4a was isolated as an impurity in a sample of a hit from a HTS campaign on 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). After separation by chiral chromatography the 4a-S, 8a-R enantiomer of compound 4a was identified as the true, potent enzyme inhibitor. The cocrystal structure of 4a with human and murine 11ß-HSD1 revealed the unique binding mode of the oxathiazine series.

Structural basis for the highly selective inhibition of MMP-13.

Inhibitors for matrix metalloproteinases (MMPs) are under investigation for the treatment of cancer, arthritis, and cardiovascular disease. Here, we report a class of highly selective MMP-13 inhibitors (pyrimidine dicarboxamides) that exhibit no detectable activity against other MMPs. The high-resolution X-ray structures of three molecules of this series bound to MMP-13 reveal a novel binding mode characterized by the absence of interactions between the inhibitors and the catalytic zinc.

A hydrocarbon ruler measures palmitate in the enzymatic acylation of endotoxin.

The ability of enzymes to distinguish between fatty acyl groups can involve molecular measuring devices termed hydrocarbon rulers, but the molecular basis for acyl-chain recognition in any membrane-bound enzyme remains to be defined. PagP is an outer membrane acyltransferase that helps pathogenic bacteria to evade the host immune response by transferring a palmitate chain from a phospholipid to lipid A (endotoxin). PagP can distinguish lipid acyl chains that differ by a single methylene unit, indicating that the enzyme possesses a remarkably precise hydrocarbon ruler.

Stability of the lactose permease in detergent solutions.

Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins. We present a quantitative analysis of the stability of the Escherichia coli lactose (lac) permease and a series of lac permease fusion proteins containing an insertion of cytochrome(b562), T4 lysozyme or beta-lactamase in the central hydrophilic loop of the permease. The stability of the proteins was evaluated under a variety of storage conditions by both a qualitative SDS-PAGE assay and by a quantitative hplc assay.

Insertion of carrier proteins into hydrophilic loops of the Escherichia coli lactose permease.

We describe the design and characterization of a set of fusion proteins of the Escherichia coli lactose (lac) permease in which a set of five different soluble "carrier" proteins (cytochrome(b562), flavodoxin, T4 lysozyme, beta-lactamase and 70 kDa heat shock ATPase domain) were systematically inserted into selected loop positions of the transporter. The design goal was to increase the exposed hydrophilic surface area of the permease, while minimizing the internal flexibility of the resulting fusion proteins in order to improve the crystallization properties of the membrane protein. Fusion proteins with insertions into the central hydrophilic loop of the lac permease were active in transport lactose, although only the fusion proteins with E.