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. By creating a bi-allelic glutamine synthetase knock out of the original CHOK1SV host cell line, they were able to improve the efficiency of generating high producing stable CHO lines for drug product manufacturing. We developed a TGE method using the same CHO K1SV GS-KO host cell line without any further cell line engineering. We also refrained from performing plasmid vector engineering. Our objective was to setup a TGE process to mimic protein quality attributes obtained from stable CHO cell line. Polyethyleneimine (PEI)-mediated transfections were performed at high cell density (4 × 10(6) cells/mL) followed by immediate growth arrest at 32 °C for 7 days. Optimizing DNA and PEI concentrations proved to be important. Interestingly, found the direct transfection method (where DNA and PEI were added sequentially) to be superior to the more common indirect method (where DNA and PEI are first pre-complexed). Moreover, the addition of a single feed solution and a polar solvent (N,N dimethylacetamide) significantly increased product titers. The scalability of process from 2 mL to 2 L was demonstrated using multiple proteins and multiple expression volumes. Using this simple, short, 7-day TGE process, we were able to successfully produce 54 unique proteins in a fraction of the time that would have been required to produce the respective stable CHO cell lines. The list of 54 unique proteins includes mAbs, bispecific antibodies, and Fc-fusion proteins. Antibody titers of up to 350 mg/L were achieved with the simple 7-day process. Titers were increased to 1 g/L by extending the culture to 16 days. We also present two case studies comparing product quality of material generated by transient HEK293, transient CHO K1SV GS-KO, and stable CHO K1SV KO pool. Protein from transient CHO was more representative of stable CHO protein compared to protein produced from HEK293.

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 = 0.0009-0.0015 µM) from a single hit compound with weak inhibitory activity (IC50 = 15 µM), discovered by high-throughput screening. Structure-based design was performed using 35 cocrystal structures of CDK2 liganded with distinct analogues of the parent compound. The profiling of compound 51 against a panel of 339 kinases revealed high selectivity for CDKs, with preference for CDK2 and CDK5 over CDK9, CDK1, CDK4, and CDK6. Compound 51 inhibited the proliferation of 13 out of 15 cancer cell lines with IC50 values between 0.27 and 6.9 µM, which correlated with the complete suppression of retinoblastoma phosphorylation and the onset of apoptosis. Combined, the results demonstrate the potential of this new inhibitors series for further development into CDK-specific chemical probes or therapeutics.

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. Pilot screening of 1453 compounds led to the discovery of 12 compounds with displacement activities (EC(50) values) ranging from 6 to 44 µM, all of which were classified as ATP-site-directed ligands. Four new type I inhibitor scaffolds were confirmed by X-ray crystallography. Although this small compound library contained only ATP-site-directed ligands, the application of this assay to large compound libraries has the potential to reveal previously unrecognized chemical scaffolds suitable for structure-based design of CDK2 inhibitors with new mechanisms of action.

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. The data suggest an unprecedented mechanism of action, in which induced-dipole forces along the Ala273 side chain alter the charge distribution of the DFG backbone, allowing the DFG to unwind. As the ADFG sequence and three-dimensional structure is highly conserved, DFG-out inhibitors of other kinases may be designed by specifically targeting the flanking alanine residue with electric dipoles.

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. CDKs are unique among protein kinases, as their functionality strictly depends on association with their partner proteins, the cyclins. In an effort to identify potential target sites for disruption of the CDK-cyclin interaction, we probed the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS) with human CDK2 and cyclin A using fluorescence spectroscopy and protein crystallography. ANS interacts with free CDK2 in a saturation-dependent manner with an apparent K(d) of 37 µM, and cyclin A displaced ANS from CDK2 with an EC(50) value of 0.6 µM. Co-crystal structures with ANS alone and in ternary complex with ATP site-directed inhibitors revealed two ANS molecules bound adjacent to one another, away from the ATP site, in a large pocket that extends from the DFG region above the C-helix. Binding of ANS is accompanied by substantial structural changes in CDK2, resulting in a C-helix conformation that is incompatible for cyclin A association. These findings indicate the potential of the ANS binding pocket as a new target site for allosteric inhibitors disrupting the interaction of CDKs and cyclins.