Development of a novel mammalian display system for selection of antibodies against membrane proteins.

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD ) as low as 0.8 nm We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule-positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.

Structural basis of quinolone inhibition of type IIA topoisomerases and target-mediated resistance.

Quinolone antibacterials have been used to treat bacterial infections for over 40 years. A crystal structure of moxifloxacin in complex with Acinetobacter baumannii topoisomerase IV now shows the wedge-shaped quinolone stacking between base pairs at the DNA cleavage site and binding conserved residues in the DNA cleavage domain through chelation of a noncatalytic magnesium ion. This provides a molecular basis for the quinolone inhibition mechanism, resistance mutations and invariant quinolone antibacterial structural features.

Baculovirus production of fully-active phosphoinositide 3-kinase alpha as a p85alpha-p110alpha fusion for X-ray crystallographic analysis with ATP competitive enzyme inhibitors.

Phosphoinositide 3-kinases have been targeted for therapeutic research because they are key components of a cell signaling cascade controlling proliferation, growth, and survival. Direct activation of the PI3Kalpha pathway contributes to the development and progression of solid tumors in breast, endometrial, colon, ovarian, and gastric cancers. In the context of a drug discovery effort, the availability of a robust crystallographic system is a means to understand the subtle differences between ATP competitive inhibitor interactions with the active site and their selectivity against other PI3Kinase enzymes. To generate a suitable recombinant design for this purpose, a p85alpha-p110alpha fusion system was developed which enabled the expression and purification of a stoichiometrically homogeneous, constitutively active enzyme for structure determination with potent ATP competitive inhibitors (Raha et al., in preparation) [56]. This approach has yielded preparations with activity and inhibition characteristics comparable to those of the full-length PI3Kalpha from which X-ray diffracting crystals were grown with inhibitors bound in the active site.

Crystal structure of human cytochrome P450 2D6.

Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds.