The Post-translational Synthesis of Hypusine In eIF5A and role of polyamines in cell growth and death

National Institute Of Dental & Craniofacial Research

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Abstract

Development of a new non-radioactive DHPS assay amenable to high throughput screening (HTS) A conventional deoxyhypusine synthase assay measures the incorporation of radioactivity from spermidine (from its aminobutyl moiety) into eIF5A protein. This method is not suitable for the HTS format. If eIF5A substrate is omitted from the DHPS reaction mixture, the enzyme engages in the abortive cycles of spermidine cleavage and the release of NADH. The NADH released from this partial reaction can be measured by coupling with the NADH-Glo assay. The non-radioactive DHPS/NADH-Glo coupled assay is highly specific, sensitive and reproducible and could be configured for HTS of small molecule libraries for the identification of new inhibitors/enhancers of DHPS. Development of a RapidFire Mass Spec-based DHPS assay (collaboration with NCATS, NIH) Drawback of the DHPS/NADH-Glo assay for HTS application is the high cost of the commercial NADH-Glo assay kit (Promega). Thus, we have undertaken a new approach to measure DHPS activity by a Mass-Spec based method. The DHPS complete or partial reaction generates 1,3-diaminopropane (DAP) as a byproduct from cleavage of spermidine. However, the MW of DAP is too small to be detected by Mass Spec. Thus, derivatives of DAP were prepared by reaction with Di-tert-butyl decarbonate (Boc2O). Boc2O derivatives displayed strong signals. This method has been optimized and will be applied to screen MIPE, NPACT and Genesis libraries (500,000 compound library) at NCATS.