Strategic Design of Catalytic Lysine-Targeting Reversible Covalent BCR-ABL Inhibitors*.

Targeted covalent inhibitors have re-emerged as validated drugs to overcome acquired resistance in cancer treatment. Herein, by using a carbonyl boronic acid (CBA) warhead, we report the structure-based design of BCR-ABL inhibitors via reversible covalent targeting of the catalytic lysine with improved potency against both wild-type and mutant ABL kinases, especially ABLT315I bearing the gatekeeper residue mutation. We show the evolutionarily conserved lysine can be targeted selectively, and the selectivity depends largely on molecular recognition of the non-covalent pharmacophore in this class of inhibitors, probably due to the moderate reactivity of the warhead. We report the first co-crystal structures of covalent inhibitor-ABL kinase domain complexes, providing insights into the interaction of this warhead with the catalytic lysine. We also employed label-free mass spectrometry to evaluate off-targets of our compounds at proteome-wide level in different mammalian cells.

Liquid formulations for stabilizing IgMs during physical stress and long-term storage.

PURPOSE: To develop a liquid formulation for IgMs to survive physical stress and storage. METHODS: Stabilizing formulations for 8 monoclonal immunoglobulin (IgMs) were found using differential scanning calorimetry (DSC). In these formulations, the IgMs were subjected to stress and storage and analyzed by size exclusion chromatography and fluorescence activated cell sorting. Structure was analyzed using small-angle X-ray scattering (SAXS). RESULTS: The highest conformational stability was found near the isoelectric point and further enhanced by addition of sorbitol, sucrose and glycine. For 2 IgMs, the pH optimum for conformational and storage stability did not correspond. Lowering the pH led to the desired storage stability. Optimized formulations prevented aggregation and fragmentation from shear stress, freeze-thaw cycles, accelerated storage and real time storage at 4°C and -20°C for 12 months. Optimized formulations also preserved immunoreactivity for 12 months. SAXS indicated that IgM in stabilizing conditions was closer to the structural IgM model (2RCJ) and less susceptible for aggregation. CONCLUSIONS: A long-term stabilizing formulation for 8 IgMs was found comprising 20% sorbitol and 1 M glycine at pH 5.0-5.5 which may have broad utility for other IgMs. Formulation development using DSC and accelerated storage was evaluated in this study and may be used for other proteins.

Liquid formulations for long-term storage of monoclonal IgGs.

Proteins like immunoglobulin (IgGs) are prone to degradation by a variety of pathways. In this study, a stabilizing formulation for long-term storage of a panel of seven monoclonal IgGs was found using differential scanning calorimetry (DSC). In the chosen formulations, the IgGs were subjected to stress, accelerated and real-time storage, and analyzed by size exclusion chromatography to determine fragment and aggregate content, and fluorescence-activated cell sorting to measure immunoreactivity. All IgGs showed the greatest conformational stability near their isoelectric point which was enhanced by adding sorbitol, sucrose, glycine, and sodium chloride. Optimized formulations, found by DSC, containing 20 % sorbitol and 1 M glycine prevented IgG aggregation and fragmentation and conserved immunoreactivity against shear stress, multiple freeze-thaw cycles, accelerated storage at 37 °C, and 12 months storage at 4 and -20 °C. Relatively poor thermal stability of the antigen-binding fragment domain was shown to limit storage stability of IgGs. This study confirms the predictive power of DSC to find storage formulations which protect IgGs during stress and long-term storage from aggregation and degradation. Liquid formulations found in this study may have a broad utility for other IgGs.