A chemical derivation MRM method for high-throughput MS-based screening to identify small-molecule modulators of deoxyhypusine synthase

Authors

• CA LeClair
• D Tao
• Y Fang
• KCC Cheng
• W Zhao
• R Tharakan
• SM Yang
• MH Park
• MD Hall

Abstract

Deoxyhypusine synthase (DHS) is required for modification and activation of hypusine within eukaryotic initiation factor 5A (eIF5A), which is involved in protein biosynthesis, cell proliferation, immune cell differentiation, and aging. Covalent binding of spermidine to a specific lysine of eIF5A is catalyzed by DHS to form a deoxyhypusine residue. Disruption of this process has been implicated in cancer, HIV replication, inflammation, and diabetes. We developed a targeted MS-based assay using multiple reaction monitoring (MRM) of reaction byproduct 1,3-diaminopropane (DAP) to identify modulators of DHS. However, the poor ionization efficiency of DAP prohibited detection at mM levels, which we improved through chemical derivatization. The successful method employed amidation of the primary amines to provide N-Boc-protected DAP (BocDAP), which significantly improved ionization efficiency providing a 1-million-fold increase in sensitivity compared to MS analysis of DAP. This protocol was modified to a single one-pot reaction within a 384-well plate format utilizing automated liquid handling for expedient high-throughput sample preparation. The assay method conditions were optimized to enable RapidFire MS-based compound screening of the LOPAC 1280 library to identify potential modulators. Data analysis of the chemical structures revealed 24 of 46 identified inhibitors (>75% inhibition rate) contained a catechol moiety with 17 contained in a fused-ring core. An HPLC-TOF analysis method was utilized to assess completion of the DHS assay. A protein peak was observed having a 71 Da increase in molecular weight compared to eIF5A indicating conversion of lysine50 to a deoxyhypusine residue, which is the first confirmation of the proposed DHS pathway.

Scientific Focus Area: Chemical Biology

This page was last updated on Tuesday, August 6, 2024