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The efficacy of therapeutic antibodies that induce antibody-dependent cellular cytotoxicity can be improved by reduced fucosylation. Consequently, fucosylation is a critical product attribute of monoclonal antibodies produced as protein therapeutics. Small molecule fucosylation inhibitors have also shown promise as potential therapeutics in animal models of tumors, arthritis, and sickle cell disease. Potent small molecule metabolic inhibitors of cellular protein fucosylation, 6,6,6-trifluorofucose per-O-acetate and 6,6,6-trifluorofucose (fucostatin I), were identified that reduces the fucosylation of recombinantly expressed antibodies in cell culture in a concentration-dependent fashion enabling the controlled modulation of protein fucosylation levels. 6,6,6-Trifluorofucose binds at an allosteric site of GDP-mannose 4,6-dehydratase (GMD) as revealed for the first time by the X-ray cocrystal structure of a bound allosteric GMD inhibitor. 6,6,6-Trifluorofucose was found to be incorporated in place of fucose at low levels (<1%) in the glycans of recombinantly expressed antibodies. A fucose-1-phosphonate analog, fucostatin II, was designed that inhibits fucosylation with no incorporation into antibody glycans, allowing the production of afucosylated antibodies in which the incorporation of non-native sugar is completely absent-a key advantage in the production of therapeutic antibodies, especially biosimilar antibodies. Inhibitor structure-activity relationships, identification of cellular and inhibitor metabolites in inhibitor-treated cells, fucose competition studies, and the production of recombinant antibodies with varying levels of fucosylation are described.

WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine-piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.

Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcription factor that acts as the master regulator of cellular response to reduced oxygen levels, thus playing a key role in the adaptation, survival, and progression of tumors. Here we report cyclo-CLLFVY, identified from a library of 3.2 million cyclic hexapeptides using a genetically encoded high-throughput screening platform, as an inhibitor of the HIF-1α/HIF-1β protein-protein interaction in vitro and in cells. The identified compound inhibits HIF-1 dimerization and transcription activity by binding to the PAS-B domain of HIF-1α, reducing HIF-1-mediated hypoxia response signaling in a variety of cell lines, without affecting the function of the closely related HIF-2 isoform. The reported cyclic peptide demonstrates the utility of our high-throughput screening platform for the identification of protein-protein interaction inhibitors, and forms the starting point for the development of HIF-1 targeted cancer therapeutics.

In biopharmaceutical process development, it is desirable to identify sites of covalent degradations to ensure product consistency. One characterization method used for therapeutic immunoglobulin gamma (IgG) 1 antibodies is limited LysC proteolysis followed by reversed-phase LC/MS. Limited LysC proteolysis leads to high efficiency cleavage at the C-terminal side of the hinge lysine 222 residue, generating Fab and Fc fragments. In this report, we show that IgG 1 samples incubated under mildly acidic conditions at elevated temperatures were partially resistant to LysC cleavage at the hinge and resulted in a species where one of the Fab arms remained connected to the Fc region (Fab-Fc). The growth of the Fab-Fc species was proportional to the duration and storage temperature of the incubation period and correlated with the amount of isomerization of the aspartic acid residue preceding lysine 222, determined by peptide mapping. The isomerization rates of samples stored for up to one year at 4 degrees C, 6 months at 29 or 37 degrees C, or 3 months at 45 degrees C were determined, and the activation energy for this conversion was calculated to be approximately 33 kJ mol(-1). The apparent isomerization rate constant was only 0.02 week(-1) for samples stored at 4 degrees C, which resulted in a modest increase from 5.1 to 6.0% isoD after twenty four weeks of storage and, hence, is not a significant concern under normal storage conditions typically used for monoclonal antibodies. However, when stored at 29 degrees C, the apparent rate constant of this reaction was found to be 0.06 week(-1) and resulted in an increase from 5.1 to 21.1% isoD after twenty four weeks of storage and is a major degradant in stressed IgG 1 antibodies.