Novel 2‐Oxoglutarate Analogues Modulate the Epigenetic Activity of the Cancer‐related Human Enzyme Aspartate/Asparagine‐β‐Hydroxylase

The human oxygenase aspartate/asparagine‐β‐hydroxylase (AspH) catalyzes posttranslational β‐hydroxylations of specific Asp/Asn‐residues in epidermal growth factor‐like domains (EGFDs) by using 2‐oxoglutarate (2OG) as a cofactor. [1] AspH is overexpressed in certain cancer cells and translocates from the endoplasmic reticulum to the cell surface membrane, a process which correlates to enhanced tumor invasiveness and poor clinical prognosis. [2] We recently reported the production of a truncated human AspH‐construct in E. coli that was catalytically active [3] and used for the development of a high‐throughput mass spectrometry‐based assay to monitor AspH‐activity in vitro . Herein, we report a novel synthesis of non‐natural 2OG analogues which were either alternative AspH‐cofactors or small‐molecule AspH‐inhibitors. Commercial starting materials were converted in three synthetic steps to previously inaccessible 2OG analogues bearing alkyl‐, aryl‐, or heteroaryl‐substituents. This novel synthesis compares favorably to literature‐reported syntheses as it features a broad substrate scope, high reaction yields, and simple reaction protocols. A diverse library of 2OG analogues (>35) was obtained and evaluated in the AspH‐assay. Potent AspH‐inhibitors were identified and ranked according to their (IC 50 )‐values, constituting the first detailed structure activity relationship study on AspH using small‐molecules with a known mode of inhibition (replacing 2OG in the active site). Several 2OG analogues could substitute 2OG as cofactor and thus promote enzymatic turnover. Michaelis constants (K M ) were determined and used to compare the efficiency of the 2OG analogues with 2OG. The selectivity of the novel AspH‐inhibitors and ‐cofactors was evaluated using structurally and functionally related human 2OG‐dependent oxygenases (i.e. PHD2, FIH, JMJD5). Finally, crystallographic experiments were performed to obtain further structural information on the substrate requirements of the AspH active site; thus enabling future studies to design selective AspH‐inhibitors as potential anticancer therapeutics. In conclusion, a novel efficient synthesis of 2OG analogues was developed providing access to important chemical tools for mechanistic and inhibition studies of 2OG‐dependent oxygenases. The relevance of this compound class was highlighted by the identification of novel potent and selective small‐molecule inhibitors of the cancer‐related human enzyme AspH. Support or Funding Information We thank the Wellcome Trust and Cancer Research UK for funding. L.B. thanks the Deutsche Forschungsgemeinschaft for a fellowship (BR 5486/2‐1).[1] Stenflo , J. ; Holme , E. ; Lindstedt , S. ; Chandramouli , N. ; Tsai Huang , L. H. ; Tam , J. P. ; Merrifield , R. B. ; Proc. Natl. Acad. Sci. USA 1989 , 86 , 444 . [2] Ince , N. ; de la Monte , S. M. ; Wands , J ; Cancer Res . 2000 , 60 , 1261 . [3] Pfeffer , I. ; Brewitz , L. ; Krojer , T. ; Jensen , S. A. ; Kochan , G. T. ; Kershaw , N. J. ; Hewitson , K. S. ; McNeill , L. A. ; Kramer , H. ; Münzel , M. ; Hopkinson , R. J. ; Oppermann , U. ; Handford , P. A. ; McDonough , M. A. ; Schofield , C. J. ; Nat. Commun . 10.1038/s41467-019-12711-7 .