A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment.

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line.

Development of highly potent and selective diaminothiazole inhibitors of cyclin-dependent kinases.

Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases that act as key regulatory elements in cell cycle progression. We describe the development of highly potent diaminothiazole inhibitors of CDK2 (IC50 = 0.0009-0.

A novel approach to the discovery of small-molecule ligands of CDK2.

In an attempt to identify novel small-molecule ligands of cyclin-dependent kinase 2 (CDK2) with potential as allosteric inhibitors, we have devised a robust and cost-effective fluorescence-based high-throughput screening assay. The assay is based on the specific interaction of CDK2 with the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS), which binds to a large allosteric pocket adjacent to the ATP site. Hit compounds that displace ANS directly or indirectly from CDK2 are readily classified as ATP site binders or allosteric ligands through the use of staurosporine, which blocks the ATP site without displacing ANS.

A novel mechanism by which small molecule inhibitors induce the DFG flip in Aurora A.

Most protein kinases share a DFG (Asp-Phe-Gly) motif in the ATP site that can assume two distinct conformations, the active DFG-in and the inactive DFG-out states. Small molecule inhibitors able to induce the DFG-out state have received considerable attention in kinase drug discovery. Using a typical DFG-in inhibitor scaffold of Aurora A, a kinase involved in the regulation of cell division, we found that halogen and nitrile substituents directed at the N-terminally flanking residue Ala273 induced global conformational changes in the enzyme, leading to DFG-out inhibitors that are among the most potent Aurora A inhibitors reported to date.

Discovery of a potential allosteric ligand binding site in CDK2.

Cyclin-dependent kinases (CDKs) are key regulatory enzymes in cell cycle progression and transcription. Aberrant activity of CDKs has been implicated in a number of medical conditions, and numerous small molecule CDK inhibitors have been reported as potential drug leads. However, these inhibitors exclusively bind to the ATP site, which is largely conserved among protein kinases, and clinical trials have not resulted in viable drug candidates, attributed in part to the lack of target selectivity.