Facile Modulation of Antibody Fucosylation with Small Molecule Fucostatin Inhibitors and Cocrystal Structure with GDP-Mannose 4,6-Dehydratase.

Abstract

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.

• Authors: John G Allen, Mirna Mujacic, Michael J Frohn, Alex J Pickrell, Paul Kodama, Dhanashri Bagal, Tisha San Miguel, E Allen Sickmier, Steve Osgood, Aleksander Swietlow, Vivian Li, John B Jordan, Ki-Won Kim, Anne-Marie C Rousseau, Yong-Jae Kim, Seb Caille, Mike Achmatowicz, Oliver Thiel, Christopher H Fotsch, Pranhitha Reddy, John D McCarter
• Journal: ACS chemical biology (ACS Chem Biol) Vol. 11 Issue 10 Pg. 2734-2743 (10 21 2016) ISSN: 1554-8937 [Electronic] United States
• PMID: 27434622 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Small Molecule Libraries
• Fucose
• Guanosine Diphosphate Mannose
• Hydro-Lyases

Topics

• Animals
• CHO Cells
• Cricetinae
• Cricetulus
• Crystallography, X-Ray
• Fucose (antagonists & inhibitors, metabolism)
• Guanosine Diphosphate Mannose (metabolism)
• Hydro-Lyases (metabolism)
• Mass Spectrometry
• Molecular Structure
• Small Molecule Libraries
• Surface Plasmon Resonance